From the Department of Pathology and Laboratory Medicine, University of Wisconsin, Madison, Wisconsin 53562-8550
Received for publication, November 4, 2002, and in revised form, February 17, 2003
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ABSTRACT |
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Fibroblast growth factor-2 (FGF2) is a potent
angiogenic factor in gliomas. Heparan sulfate promotes ligand binding
to receptor tyrosine kinase and regulates signaling. The goal of this
study was to examine the contribution of heparan sulfate proteoglycans (HSPGs) to glioma angiogenesis. Here we show that all brain endothelial cell HSPGs carry heparan sulfate chains similarly capable of forming a
ternary complex with FGF2 and fibroblast growth factor receptor-1c and
of promoting a mitogenic signal. Immunohistochemical analysis revealed
that glypican-1 was overexpressed in glioma vessel endothelial cells,
whereas this cell-surface HSPG was consistently undetectable in normal
brain vessels. To determine the effect of increased glypican-1
expression on FGF2 signaling, we transfected normal brain endothelial
cells, which express low base-line levels of glypican-1, with this
proteoglycan. Glypican-1 expression enhanced growth of brain
endothelial cells and sensitized them to FGF2-induced mitogenesis
despite the fact that glypican-1 remained a minor proteoglycan. In
contrast, overexpression of syndecan-1 had no effect on growth or FGF2
sensitivity. We conclude that the glypican-1 core protein has a
specific role in FGF2 signaling. Glypican-1 overexpression may
contribute to angiogenesis and the radiation resistance characteristic
of this malignancy.
High-grade gliomas are characterized by a highly aggressive
clinical course, active angiogenesis, and endothelial cell
proliferation. Fibroblast growth factor-2
(FGF2)1 is one of the most
potent stimulators of angiogenesis in wound healing and tumor growth.
FGF2 induces all the important components of blood vessel growth,
including degradation of the extracellular matrix, endothelial cell
migration and proliferation, and differentiation into vascular tubes.
FGFs signal through transmembrane receptor tyrosine kinases, the FGF
receptors (FGFRs) (1). Of the four known FGFRs, FGFR1 is the one most
prevalently found on endothelial cells (2).
Stable binding of the FGF ligand to the receptor tyrosine kinase and
sustained signaling require the presence of heparan sulfate (HS)
glycosaminoglycans (3, 4). The participation of HS polysaccharides in
the ternary FGF receptor signaling complex is facilitated
through HS-binding motifs on both the FGF ligand and the receptor
tyrosine kinase (5). HS chains are characterized by complex sulfation
patterns resulting in distinct protein-binding domains, although it is
currently unclear how the assembly of such domains is regulated.
Experimental evidence indicates that stimulatory as well as inhibitory
moieties are embedded within HS chains and that their relative balance
determines the net effect on FGF signaling (6). Cells have the ability
to rapidly adjust their HS to respond to a changing microenvironment
(7). HS proteoglycan (HSPG) alterations have also been reported to
occur during malignant progression. Recently, we described HS
alterations in breast carcinomas, resulting in increased FGF2 binding
and enhanced ternary FGF receptor complex assembly (8).
In vivo, the vast majority of HS exists in covalent linkage
to core proteins, the HSPGs. HSPGs can be divided into cell-surface forms (syndecans and glypicans) and secreted extracellular matrix forms
(e.g. perlecan). Literature reports on which HSPGs promote FGF2 signaling are conflicting. One study identified perlecan as the
sole stimulatory HSPG, whereas syndecans and glypicans were found to
inhibit FGF signaling (9). Others reported that syndecans and glypicans
also promote FGF2 signaling (10). These divergent results suggest that
HSPGs with different core proteins assume this role depending on cell
type and functional context. The question of whether different HSPG
core proteins produced by one cell type can carry HS chains with
different effects (stimulatory versus inhibitory) on FGF2
signaling is currently unresolved.
Published reports on the role of HSPGs in angiogenesis are
sparse (11). Quiescent as well as angiogenically active blood vessels
contain ample amounts of FGF2. This suggests that FGF2-mediated angiogenesis is regulated by additional factors such as HSPGs. Perlecan
has been described as a pro-angiogenic molecule (12) as well as an
inhibitor of FGF2 signaling in blood vessels (13). Other investigators
have attributed an exclusive role to syndecan-4 in FGF2-induced
angiogenesis and ascribed a special role to the core protein of this
HSPG (14).
The goal of this study was to examine the contribution of different
brain endothelial cell HSPGs to FGF2 signaling and glioma angiogenesis.
We found that despite the fact that all endothelial cell HSPGs have the
potential to promote binding of FGF2 to FGFR1c through their HS chains
and to promote signaling, specific HSPGs appear to carry out the
function of principal FGF2 co-receptors in tumor angiogenesis.
Specifically, glypican-1 is dramatically up-regulated in glioma
vessels, leading to enhanced growth and endothelial sensitivity to FGF2 stimulation.
Reagents--
Yeast-derived human recombinant FGF2 was kindly
provided by Dr. Brad Olwin (University of Colorado, Boulder, CO).
Anti-syndecan-1 antibody (B-B4) was purchased from Serotec (Raleigh,
NC). Antibodies to syndecan-2 (10H4) (15), syndecan-3 (1C7) (16),
syndecan-1 and -3 (2E9) (16), syndecan-4 (8G3) (17), and glypican-1
(S1) (18) were a generous gift from Dr. Guido David (University of Leuven, Leuven, Belgium). Rabbit polyclonal anti-syndecan-4 antibody was a gift from Dr. Alan Rapraeger (University of Wisconsin) (19). Neomarkers anti-perlecan antibody was purchased from LabVision (Freemont, CA), and anti- Cell Culture and FGF2 Response Assays--
Dr. Robert Auerbach
(University of Wisconsin, Madison, WI) kindly provided mouse brain
endothelial (MBE) cells (21). GM7373 immortalized bovine endothelial
cells were obtained from the Coriell Institute for Medical Research
(Camden, NJ) (22). Both cell types were grown in Dulbecco's modified
Eagle's medium supplemented with 10% calf serum. Human umbilical vein
endothelial cells (HUVECs) and human dermal microvascular endothelial
cells (HMVECs) were from Cambrex Corp. (East Rutherford, NJ).
Human saphenous vein endothelial cells (HSVECs) were purchased from VEC
Technologies (Rensselaer, NY). All human primary endothelial cells were
grown on gelatin-coated tissue culture dishes in EGM-2
endothelial cell growth medium (Cambrex Corp.). Proliferation of
adherent cells was measured with a bromodeoxyuridine (BrdUrd)
incorporation assay (BrdUrd colorimetric, Roche Diagnostics) according
to the manufacturer's instructions. The cells were starved in
serum-free medium for 24 h and then treated with FGF2 for 24 h with BrdUrd label added for the final 4 h. The urokinase assay
was adapted from Presta et al. (22). Briefly, GM7373 cells
were treated with FGF2 for 24 h. Cell lysates were prepared by
sonication. Ten µg of protein from cell lysate were incubated with
bovine plasminogen (40 µg/ml) and Spectrozyme PL chromogenic
substrate (330 µM). Absorbance at 405 nm was measured at
10-min intervals. Human high-molecular mass urokinase served as a
standard. All urokinase assay reagents were purchased from American
Diagnostica, Inc. (Greenwich, CT). FR1c-11 cells (BaF3 cells
transfected with the FGFR1c isoform; kindly provided by Dr. David
Ornitz, Washington University, St. Louis, MO) were maintained in RPMI
1640 medium supplemented with 10% calf serum and 10% WEHI-3
cell-conditioned medium as source of interleukin-3. For the growth
assays, cells (20,000/well) were added to 96-well tissue culture plates
in RPMI 1640 medium lacking interleukin-3 and incubated with various
combinations of FGF2 and HSPGs for 72 h. The tetrazolium
compound-based CellTiter 96 aqueous cell viability/proliferation assay
(Promega, Madison, WI) was performed according to the manufacturer's
instructions at the end of the incubation period.
Cell Transfections--
A 2.5-kb mouse glypican-1 cDNA
fragment (a generous gift from Dr. Guido David) was cloned into the
mammalian expression vector pcDNA3.1/Myc-HisA (Invitrogen). A
1.3-kb mouse syndecan-1 cDNA sequence was cloned into the pIRESneo3
mammalian expression vector (Clontech) under the
control of the cytomegalovirus promoter/enhancer. The internal ribosome
entry site in this vector permits simultaneous expression of syndecan-1
and the selection marker neomycin phosphotransferase (Neo) from one
mRNA. This vector was chosen because measurable syndecan-1
overexpression could not be achieved with the pcDNA3.1 vector. MBE
cells were stably transfected with either construct or the respective
empty vector using TransIT-LT1 transfection reagent (Mirus, Madison,
WI). After selection with G418 (500 µg/ml), cell extracts were
subjected to HSPG Western blot analysis with antibody 3G10 to determine
the levels of HSPG core proteins. Proliferation assays were carried out
on mixed transfected populations to avoid clonal selection bias.
HSPG Extractions and Complex Precipitations--
Total
endothelial cell HSPGs were purified as described (8, 23). For
selective isolation of cell-associated and extracellular matrix HSPGs,
we modified a procedure described by Elkin et al. (24).
Cells were grown for 3 days in 150-mm dishes. After rinsing, the cell
monolayer was dissolved in cold cell lysis buffer (20 mM
NH4OH and 0.5% (w/v) Triton X-100 in phosphate-buffered
saline, pH 10) for 1.5 min. After removal of the lysate, the remaining extracellular matrix layer was washed four times with Hepes-buffered saline and extracted with 3 ml of buffer containing 10 mM
Tris, 8 M urea, 0.1% (w/v) Triton X-100, 1 mM
Na2SO4, 1 mM
phenylmethylsulfonyl fluoride, and 1 mM
N-ethylmaleimide, pH 8.0, for 5 min on ice and scraped into
a polypropylene bottle. All extractions were enriched for proteoglycans
by DEAE ion-exchange chromatography as described (8). HSPGs were
quantified by dot blotting after heparitinase digestion and stained
with antibody 3G10 using commercially available HSPGs (Sigma) as standards.
To examine the ability of HSPGs to bind to FGF2, the growth factor was
biotinylated while bound to heparin-agarose to protect its HS-binding
site and then immobilized on streptavidin-agarose beads. MBE cell HSPGs
were incubated with immobilized FGF2 and eluted with 2 M
NaCl. Fractionation of HSPGs according to their ability to promote
binding of FGF2 to FGFR1 was performed as described (8). All HSPG
preparations were digested with heparitinase and chondroitinase to
remove all glycosaminoglycan chains prior to analysis on a 3.5-15%
(w/v) Tris borate-polyacrylamide gradient gel. The membranes were
stained with anti- FGF·Receptor Complex Reconstitution in
Situ--
Residual human tissue was obtained fresh from the surgical
pathology laboratory, embedded in OCT compound (Sakura,
Torrance, CA), snap-frozen, and stored at Immunohistochemistry--
Syndecan-1 and -4 and glypican-1
staining was performed on paraffin-embedded tissues as described (8).
Staining for syndecan-2 and -3 was carried out on cryostat sections
because antibodies 10H4 and 1C7 did not produce a detectable signal on
paraffin sections. In addition, cryosections were stained for
glypican-1 with antibody S1 to confirm the paraffin section results.
Antibodies 10H4 (10 µg/ml), 1C7 (10 µg/ml), and S1 (5 µg/ml) were
detected with Alexa 546-conjugated secondary antibody. Rabbit
polyclonal anti-vWF antibody was used to localize endothelial cells in
tissue sections (see above). All fluorescence microscopy was done using
an Olympus BX51 microscope equipped with a SPOT RT slider chilled CCD
digital camera (Diagnostic Instruments, Sterling Heights, MI).
Statistics--
Growth responses at different concentrations
were compared using the Wilcoxon rank sum test. Correlations between
staining results were examined by regression analysis using the
StatView statistics program.
Heparan Sulfate Is Required for FGF2 Signaling in Endothelial
Cells--
A number of studies on a variety of cell types have
demonstrated that HS is necessary for stable binding of FGF2 to FGFRs and for signaling. To establish an HS requirement for FGF2 signaling in
endothelial cells, we evaluated the effect of abolishing HS on the key
angiogenic and FGF2-stimulated events mitogenesis and urokinase
production. MBE cells show a robust proliferative response to FGF2. In
GM7373 bovine endothelial cells, FGF2 prominently induces
urokinase-type plasminogen activator, a crucial enzyme responsible for
extracellular matrix degradation during angiogenesis (27). Experimental
evidence points toward divergent signaling pathways involved in the
regulation of these two cellular responses (22). Functional HS was
abolished by treatment with sodium chlorate, a competitive inhibitor of
glycosaminoglycan sulfation (28). Chlorate treatment resulted in a
significant inhibition of MBE cell proliferation (Fig.
1A) and complete suppression
of urokinase production by GM7373 bovine endothelial cells (Fig.
1B). Importantly, these responses were recovered in either
cell type when HS sulfation was restored with sodium sulfate or by
providing soluble heparin as an HS substitute to the cells in
culture.
All Endothelial Cell HSPGs Are Capable of Acting as FGF2
Co-receptors--
HS exists almost exclusively in covalent linkage to
a heterogeneous group of core proteins. As a first step in determining which HSPGs participate in the FGF2 co-stimulator role, we measured HSPG expression in different endothelial cells. Microvascular MBE
cells, primary HMVECs, and human umbilical and saphenous vein endothelial cells were analyzed by Western blotting. Removal of all
glycosaminoglycan chains by treatment with heparitinase and chondroitinase allowed the detection of discrete bands on
SDS-polyacrylamide gradient gels. Visualization of all HSPGs was
possible using an antibody (clone 3G10) directed against the HS
stubs (anti-
FGF2-binding ability would be expected to be the minimal requirement
for HSPGs to function as FGF2 co-stimulators. To detect any potential
differences in FGF2 binding between endothelial HSPGs, we incubated
purified MBE cell HSPGs with immobilized FGF2 and analyzed
the HSPGs found in this complex. The spectrum of HSPGs that bound
to FGF2 (Fig. 2B, lane 3) was
similar to that of the loading control (lane 1), indicating
that all HSPG core proteins are decorated with FGF2-binding HS chains.
No binding was detected when beads lacked FGF2 (Fig. 2B,
lane 5) or when heparin was added to the binding reaction as
a competitive inhibitor (lane 7).
FGF2 binding is likely necessary for HSPGs to stimulate signaling, but
is clearly not sufficient. The ternary receptor complex, which is
generally regarded as the active signaling complex, is expected to
require additional HS motifs interacting with HS-binding sites on FGFR
(5). Because FGFR1 is reportedly the only FGFR expressed by MBE cells
(2), and FGFR1c the most prevalent FGFR1 isoform,2 HSPGs isolated from
this cell type were incubated with immobilized FGFR1c in the presence
of FGF2. HSPGs participating in this ternary complex were analyzed on
gradient gels (Fig. 2C, lane 4). Again, the
relative abundance of HSPGs found in the complex was similar to that
found in the loading control (Fig. 2C, lane 1),
suggesting that all core proteins carry HS chains with a similar
potential to promote FGF2 binding to FGFR1c. Complex formation was
abolished by omitting FGF2 from the reaction (Fig.
2C, lane 3) or by digesting the HSPGs with
heparitinase prior to binding (lane 2), demonstrating the
requirement for the FGF2 ligand and for intact HS chains. The complexes
were similarly resistant to a NaCl concentration of 600 mM,
but dissociated progressively when exposed to NaCl concentrations
between 1 and 1.4 M (Fig. 2C, lanes
5 and 6). To exclude the possibility that FGF2 bound to
recombinant FGFR1c independent of heparan sulfate in these experiments,
we immobilized biotinylated FGF2 on streptavidin-coated plates and
measured binding of FGFR1c. As expected, FGFR1c binding to immobilized
FGF2 was highly dependent on the presence of heparin (data not shown). In summary, this series of in vitro experiments demonstrates
that HS chains on all MBE cell HSPGs are similarly capable of binding FGF2 and of promoting binding of FGF2 to FGFR1c in a ternary complex.
Different Classes of Brain Endothelial Cell HSPGs Have a Similar
Ability to Promote FGF2 Signaling through FGFR1c--
The formation of
a high-affinity ternary FGF·receptor complex does not
necessarily assure signal generation. It is possible that a dimerized
receptor signaling complex requires additional structural features
within HS not examined in the assays used so far. To examine potential
differences in the ability of endothelial cell HSPGs to promote
receptor activation, HSPGs were fractionated into functional classes
using selective extraction protocols (adapted from Ref. 24). The
relative purity of cell-associated HSPGs (composed primarily of
syndecans and glypican-1) and extracellular matrix HSPGs (composed
primarily of perlecan) was tested by Western blot analysis (Fig.
3A). Purified HSPGs were
carefully quantified by dot blotting using antibody 3G10 (data not
shown). Their activity was tested using FR1c-11 cells, which are BaF3
murine lymphoid cells transfected with FGFR1c (3). FR1c-11 (and parent
BaF3) cells lack endogenous HSPGs and therefore depend on an exogenous source of HSPGs for FGF2-induced mitogenesis. HSPG dose-response curves
in the presence or absence of FGF2 did not differ significantly between
cell-associated, extracellular matrix, and total HSPGs, indicating that
HS chains on these different core proteins are similarly capable of
promoting an FGF2 signal (Fig. 3B).
Human Glioma Vessel HSPGs Have an Increased Ability to Form the
Ternary FGF2·HSPG·FGFR1c Complex Compared with Normal Brain Vessel
HSPGs: Glypican-1 Is Overexpressed in Human Glioma Vessels--
To
examine whether vascular HSPG binding activities are altered in
gliomas, we reconstituted the FGF2·HSPG·FGFR1 complex in situ on frozen or paraffin sections of human tissue samples.
Biopsy samples from grade IV astrocytomas (glioblastoma multiforme) and non-neoplastic brain controls (temporal lobectomy samples and normal
white matter adjacent to tumors) were compared. In these experiments,
binding of soluble FGFR1c to HSPGs within the tissue was measured in
the presence of FGF2. FGFR1c binding was notably increased in glioma
vessels (Fig. 4, A and
E) compared with normal brain vessels (Fig. 4, D
and H) in 7 of 11 cases examined (64%). No recognizable
morphologic features distinguished FGFR1c "binding" from
"non-binding" cases. The binding signal was abolished by enzymatic
degradation of HS with heparitinase (Fig. 4, B and
F), demonstrating HS dependence, or by omitting the FGF2
incubation step (Fig. 4, C and G). This result
indicates either a qualitative or quantitative alteration of glioma
vessel HSPGs in the majority of tumors. Paraffin sections stained with
anti-HS stub antibody 3G10 demonstrated an increase in total HSPG
content in glioma vessels compared with normal brain vessels (Fig.
5, A and B), suggesting that increased vascular HSPG content rather than altered HS
structure is responsible for the elevated binding activity. Regression
analysis of individual cases revealed a significant association between
FR1-AP binding and antibody 3G10 staining intensity (p = 0.047).
Next, we investigated whether the increased binding activity of glioma
vessel HSPGs is accompanied by an increase in any particular HSPG core
protein. Immunohistochemical analysis of paraffin-embedded human
tissues revealed that syndecan-1 was undetectable in normal and glioma
vessels (Fig. 5H). Low levels of syndecan-4 were seen in
both glioma (Fig. 5D) and normal brain (Fig. 5C)
vessels. In contrast, glypican-1 was strikingly overexpressed in glioma
vessel endothelial cells (moderate-to-strong staining in five of eight cases, 63%) (Fig. 5F), whereas normal brain vessel
endothelial cells consistently lacked this cell-surface HSPG (Fig.
5E).
The study of human tissue samples was expanded to include cryosections.
The purpose of these additional labeling experiments was to measure
expression of HSPGs undetectable in paraffin sections, to confirm the
striking glypican-1 expression pattern seen in the paraffin sections,
and to examine co-localization of HSPGs with endothelial cells in more
detail. Cryosections were stained with antibodies to syndecan-2 and -3 and glypican-1 (red fluorescence channel). Dual labeling was performed
on the same sections with antibody to vWF to identify endothelial cells
(green fluorescence channel). When examining the cryosections, blood
vessels were initially identified viewing the green channel, and then
HSPG expression was evaluated by switching to the red channel.
Syndecan-2 was mildly increased in glioma vessel endothelial cells
(Fig. 6, A and E)
compared with normal brain vessels, where this HSPG was expressed only
in low amounts (Fig. 6, I and M). In addition, syndecan-2 was abundantly present in a fibrillar and linear pattern external to tumor vessel endothelial cells, i.e. not
directly co-localizing with the vWF signal (Fig. 6E and
inset). The cellular origin of syndecan-2 in this location
is not clear. Syndecan-3 was moderately expressed in both glioma (Fig.
6, B and F) and normal brain (Fig. 6,
J and N) vessels. As in paraffin sections, glypican-1 was abundant in tumor vessel endothelial cells (Fig. 6,
C and G), whereas normal brain vessel endothelial
cells essentially lacked this HSPG (Fig. 6, K and
O). In summary, the immunohistochemistry data are consistent
with the hypothesis that increased glypican-1 is responsible for
elevated total HSPG levels in glioma vessels, but we cannot exclude the
possibility that syndecan-2 and/or HSPGs not included in the analysis
also contribute.
Glypican-1 Overexpression Stimulates Endothelial Cell Growth and
Sensitizes Endothelial Cells to FGF2 Stimulation--
MBE cells
expressed low levels of glypican-1, mirroring normal brain vessel
endothelial cells in vivo. Nevertheless, MBE cell glypican-1
was capable of supporting sustained binding of FGF2 to FGFR1 (Fig.
2C). To determine how glypican-1 up-regulation as it occurs
in tumor vessels affects FGF2 signaling in the same cell, we measured
FGF2-induced proliferation in MBE cells transfected with glypican-1.
Modest glypican-1 overexpression did not affect the levels of other
HSPG core proteins (Fig. 7A);
and importantly, even in the transfected cells, glypican-1 remained a
minor constituent of the total HSPG spectrum. Yet cells transfected
with glypican-1 displayed a dramatically elevated base-line
proliferation rate (Fig. 7B) and an increased sensitivity to
FGF2 stimulation, resulting in a leftward shift of the
dose-response curve by at least an order of magnitude (Fig.
7C). Estimated half-maximal stimulation occurred at 23.6 pM FGF2 in the mock-transfected cells and at 0.97 pM in the glypican-1-transfected cells.
To determine whether the effect of glypican-1 on MBE cell growth and
FGF2 response is specific to this core protein, MBE cells were also
transfected with syndecan-1. This HSPG was chosen because it represents
a different class of cell-surface HSPGs (transmembrane versus lipid anchor), is present at similarly low base-line
levels as glypican-1, and is not up-regulated in glioma vessels.
Similar to glypican-1-transfected cells, syndecan-1 was overexpressed at modest levels in the transfected cell population (Fig.
7D). In contrast to glypican-1-transfected cells,
syndecan-1-overexpressing cells demonstrated a slightly decreased
growth rate (Fig. 7E), and their sensitivity to FGF2-induced
mitogenesis was unchanged compared with mock-transfected controls (Fig.
7F). These results suggest a specific role for the
glypican-1 core protein in endothelial cell growth and FGF2-induced
mitogenesis independent of HS chain composition.
In summary, we have shown that all endothelial cell HSPG core
proteins carry HS chains similarly capable of binding FGF2, stabilizing
the ternary receptor signaling complex with FGFR1c, and activating this
receptor tyrosine kinase. Nevertheless, we found that glypican-1, which
is overexpressed in glioma vessels, has a specific ability to sensitize
brain endothelial cells to mitogenic FGF2 stimulation.
Vascular endothelial growth factor (VEGF) has received most of the
attention recently as a mediator of tumor angiogenesis because of its
target cell specificity, but FGF2 is similarly potent. In fact, VEGF
and FGF2 can act synergistically (29) or have distinct roles in tumor
development by exerting their angiogenic activity at different tumor
growth stages and in different tumor regions. FGF2 expression
predominates in small tumors and at the tumor periphery, whereas VEGF
expression is most prominently observed in larger tumors and in the
tumor center (30, 31), consistent with transcriptional regulation of
VEGF via hypoxia-inducible factors.
Our observation that different HSPGs produced by one cell type have
similar activities is novel and in apparent conflict with the presently
prevailing view that HSPGs are specific FGF co-receptors. Yet most
studies to date have focused on individual HSPGs and not compared
binding characteristics of the entire HSPG spectrum produced by a
single cell type. Using an affinity chromatography approach similar to
ours, investigators recently identified syndecan-1 as the HSPG
responsible for FGF receptor binding in prostate epithelial cells, suggesting that this core protein is specifically endowed with
HS chains particularly capable of interacting with the receptor tyrosine kinase (32). However, the findings of that study could also be
explained by a predominance of syndecan-1 in this cell type and
therefore in the starting material. Our data suggest that the enzyme
machinery in the Golgi responsible for HS synthesis and modification
does not distinguish between different core proteins. This does not
imply that HS chain composition is static. HS binding activities have
been shown to change dramatically over time during development and
malignant transformation (7). Also, different cell types can decorate
the same HSPG core protein with very diverse HS chains (33).
Glypican-1 overexpression in endothelial cells results in significantly
enhanced growth and improved FGF2 response despite the fact that its HS
chains are apparently not unique and the total HSPG content is not
substantially increased. This observation suggests a specific role for
the glypican-1 core protein in angiogenic FGF2 signaling and raises the
question of what distinguishes glypican-1, the sole endothelial
glypican (34), from other cell-surface HSPGs. Glypican HS chain
attachment sites are located near the cell membrane, whereas HS
attachment sites in syndecans are near the N terminus of the variably
sized ectodomain. The proximity of the HS chains to the cell surface
may support the formation of a ternary receptor complex with FGFR.
Glypicans also distinguish themselves from syndecans by their cell
membrane anchorage via glycosylphosphatidylinositol. The lack of a
transmembrane domain and the presence of a lipid tether likely endow
glypican-1 with increased mobility within the lipid bilayer, favoring
FGFR complex formation and oligomerization possibly within
cholesterol-rich lipid rafts. Interestingly, tethering FGF3 directly to
the cell surface via a glycosylphosphatidylinositol anchor potentiates its transforming activity, apparently enabling this FGF to bypass glypican binding (35).
Volk and co-workers have identified a specific role for the syndecan-4
core protein in FGF2 signaling (14). Glypican-1 could not substitute
for syndecan-4 in the HSPG-deficient cells used in this study.
Experiments with HSPG core protein chimeras and dominant-negative
approaches ascribed a unique function and possibly direct signaling
role to the syndecan-4 cytoplasmic domain. The MBE cells used in our
study produce ample endogenous HSPGs, including syndecan-4, thereby
meeting the requirement for this crucial signal. Endogenous MBE cell
HSPGs together with FGFR1 are sufficient to mediate an FGF2 response,
but apparently the signal is not optimal, as glypican-1 overexpression
further sensitizes MBE cells to respond to lower FGF2 concentrations.
The "optimal" HSPG co-receptor glypican-1 may compensate in brain
endothelial cells for suboptimal FGF2 supply and/or HSPG composition
during active angiogenesis (36). Angiogenesis modulation by HSPGs
apparently varies in different organ sites, as we observed primarily
syndecan-4 overexpression in breast carcinoma microvessels and both
glypican-1 and syndecan-4 up-regulation in skin wound granulation
tissue.2 This finding re-enforces the well established
concept of endothelial heterogeneity.
Both activation and inhibition of angiogenesis by glypican-1 apart from
FGF2 signaling have been described. Shed glypican-1 can protect
VEGF165 from oxidative damage in a chaperone-like function
(34). Glypican-1 has also been identified as an essential low-affinity
co-receptor for the potent angiogenesis inhibitor endostatin (37). The
roles for glypican-1 in neoplasia extend beyond angiogenesis.
Glypican-1 is overexpressed in pancreatic carcinomas and is required
for FGF2 signaling in this tumor cell type (38). Recently, glypican-1
overexpression has been described for breast carcinomas as well (39).
Although regulation of syndecan expression has been well studied (40),
relatively little is known about the regulation of glypican expression.
Gene silencing by promoter methylation has been reported to result in
suppression of glypican-3 expression (41). Further work will be needed
to address the signals involved in inducing glypican-1 expression in
glioma vessels.
Glypican-1-mediated sensitization of endothelial cells to FGF2 likely
contributes to the active angiogenic phenotype characteristic for
high-grade gliomas. Overexpression of this HSPG may have another deleterious effect in this grim disease. Radiation tissue injury is
mediated primarily by apoptosis of vascular endothelial cells (42).
FGF2 is a potent endothelial cell survival factor during irradiation
(42). Potentiation of this survival signal by glypican-1 may be
partially responsible for radiation resistance inevitably developing in
gliomas. The central role of glypican-1 in signaling of both FGF2 and
VEGF may, on the other hand, present an attractive opportunity for
therapeutic intervention.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
HS antibody (3G10) (20) was from Seikagaku America (Falmouth, MA). This antibody reacts with unsaturated uronate/HS "stubs" generated by heparitinase treatment. Polyclonal rabbit anti-von Willebrand factor (vWF) antibody was purchased from
Dako Corp. (Carpinteria, CA). This antibody reacts with the Factor
VIII·vWF macromolecular complex.
HS antibody 3G10 or anti-core protein antibodies
(see above). A horseradish peroxidase-conjugated secondary antibody and
SuperSignal West Femto maximum sensitivity substrate (Pierce) were used
for chemiluminescent detection.
70 °C. Binding assays
were carried out essentially as described previously (25, 26). Briefly, the sections were first incubated with FGF2 (10 nM) and
then with the soluble recombinant FGFR1c extracellular domain linked to alkaline phosphatase (referred to as FR1-AP; 30 nM) (3).
Immobilized FR1-AP was detected with monoclonal anti-AP antibody
(Sigma) and visualized with Alexa 546-conjugated donkey anti-mouse
antiserum (Molecular Probes, Inc., Eugene, OR). Rabbit polyclonal
anti-vWF antiserum (1:2000) was used to localize endothelial cells in
tissue sections, and 4,6-diamidino-2-phenylindole was applied as a
nuclear counterstain.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
HS requirement for FGF2 signaling in
endothelial cells. Endothelial cell lines were grown for 48 h
in sulfate-free medium supplemented with sodium chlorate (30 mM) to abolish sulfation of HS. After 24 h of serum
starvation, the cells were treated with FGF2 (100 pM) for
24 h in the presence or absence of SO4 (10 mM) or heparin (1 µM) as indicated.
A, proliferation of MBE cells measured by BrdUrd
incorporation. Results are expressed as -fold stimulation over
base-line growth. Bars indicate the means of six
measurements. Error bars indicate S.E. B,
urokinase-type plasminogen activator (uPA) production by
GM7373 bovine endothelial cells. Urokinase was measured with a two-step
chromogenic enzyme assay in cell lysates standardized according to
protein concentration. Bars indicate means of triplicates.
Error bars indicate S.E.
HS) remaining on the core proteins after
heparitinase digestion (Fig. 2A, lanes 1-4)
(20). The signal generated with this antibody would be expected to be a
function of the amount of core protein and the number of HS attachment
sites per molecule. However, the possibility of biased heparitinase
activity or 3G10 reactivity against different HSPGs cannot entirely be
excluded. With this semiquantitative method, we estimated that
syndecan-2 and -4 were the most prevalent cell-surface HSPGs produced
by all of the endothelial cell types examined, whereas syndecan-1 and
-3 and glypican-1 represented minor HSPG fractions in some cell types
or were undetectable in others. A relatively strong band representing
the secreted extracellular matrix HSPG perlecan was also observed in
all cells examined. The identity of most bands was established in MBE
cell extracts using core protein-specific antibodies (Fig.
2A, lanes 5 and 7-9). Glypican-1 was
identified in HMVEC extracts (Fig. 2A, lane 6)
for lack of a mouse-reactive antibody. We noted a slightly delayed
migration of human syndecan-4 (Fig. 2A, lanes 2-4) compared with murine syndecan-4 (lane 1), likely
due to the species difference. Overall, MBE cells produced the largest
amount of HSPGs. Syndecan-2 was the predominant HSPG in microvascular cells (MBE cells and HMVECs), whereas syndecan-4 predominated in large
vein endothelial cells (human umbilical and saphenous vein endothelial
cells).
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Fig. 2.
Characterization of endothelial cell HSPGs as
FGF2 co-receptors. A, spectrum of HSPGs produced by
different endothelial cell types. HSPGs extracted from different
endothelial cell types were treated with heparitinase and
chondroitinase (see "Materials and Methods") to degrade
glycosaminoglycan chains and then analyzed by SDS-PAGE using a
3.5-15% (w/v) gradient gel. Lanes 1-4, 5.0 × 105 cell equivalents stained with anti-HS stub (anti- HS)
antibody 3G10, which allows the detection of all HSPGs; lanes
5-9, 5.0 × 105 cell equivalents stained with
anti-core protein antibodies as indicated. Antibody 2E9, used for
staining of lane 8, reacted with the cytoplasmic domains of
both syndecan-1 and -3 (see "Materials and Methods" for details).
Lanes 5 and 7-9 contained extracts from MBE
cells, and lane 6 contained extracts from HMVECs.
HUVEC, human umbilical vein endothelial cells;
HSVEC, human saphenous vein endothelial cells;
Perl, perlecan; Glyp-1, glypican-1;
Synd-2, syndecan-2; Synd-1, -3, syn- decan-1 and -3; Synd-4, syndecan-4. B,
fractionation of brain endothelial cell HSPGs according to their
ability to bind FGF2. HSPGs isolated from MBE cells (1.2 × 106 cell equivalents) were incubated with FGF2-coated
agarose beads. Bound material was eluted with 2 M NaCl.
After concentration on DEAE beads, the complex eluate (C)
and supernatant (S) were analyzed as described for
A. The membrane was probed with anti-
HS antibody 3G10. A
total HSPG (T) loading control (lane 1), a
beads-only (without FGF2) control (lanes 4 and
5), and competition with heparin (1 mg/ml; lanes
6 and 7) are shown as indicated. C,
fractionation of brain endothelial cell HSPGs according to their
ability to promote binding of FGF2 to FGFR1. In the presence of FGF2
(16 nM), HSPGs isolated from MBE cells (1.2 × 106 cell equivalents) were incubated with FR1-AP fusion
protein immobilized on agarose beads. After washing with NaCl
(concentrations as indicated) and digestion with heparitinase and
chondroitinase, HSPGs complexed to the beads were analyzed by SDS-PAGE,
blotting the membrane with anti-
HS antibody (lanes 4-6).
A total HSPG (T) loading control (5.0 × 105 cell equivalents) is shown (lane 1).
Negative controls included digestion of the HSPG preparation with
heparitinase prior to complex formation (lane 2) and
omission of FGF2 from the binding reaction (lane 3).
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Fig. 3.
Activity of different brain endothelial cell
HSPGs as promoters of FGF2 signaling. Total,
cell-associated (CA), and extracellular matrix
(ECM) HSPGs were selectively extracted from MBE cell
cultures (see "Materials and Methods" for details). A,
the relative purity of the preparations was tested by Western blot
analysis using anti- HS antibody 3G10 for detection. Note that
cell-associated HSPGs were greatly enriched for syndecan-1, -2, -3, and
-4 and contained only small amounts of perlecan. In contrast,
extracellular matrix HSPGs consisted almost exclusively of perlecan.
B, total and selectively extracted HSPG preparations were
carefully quantified, and the concentrations were adjusted. Increasing
concentrations of HSPGs were added to FR1c-11 cells (HSPG-deficient
murine lymphoid cells transfect with FGFR1c) in the presence and
absence of FGF2 (10 nM). Cell number was determined after
72 h using a tetrazolium compound-based assay.
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Fig. 4.
Reconstitution of the FGF2·HSPG·FGFR1
complex in situ. Frozen sections of glioma tissues
were first incubated with FGF2 (10 nM) and after a washing
step with soluble FR1-AP fusion protein (30 nM). In this
assay, the tissue section served as a source of HSPGs. Bound FR1-AP was
detected by immunofluorescence using an anti-AP antibody followed by
Alexa 546-conjugated secondary antibody (red channel). A and
E, FR1-AP binding in high-grade glioma vessels. B
and F, negative control for FR1-AP binding in high-grade
glioma vessels. The tissue section was treated with heparitinase
(Hep'ase) prior to the FGF2 binding step to identify the
tissue binding sites as HSPGs. C and G, negative
control for FR1-AP binding in high-grade glioma vessels. The FGF2
incubation step was omitted to demonstrate FGF2 dependence of FR1-AP
binding. D and H, FR1-AP binding in normal brain
vessels. The granular red stain in the brain parenchyma
represents autofluorescence. E-H are merged images in which
endothelial cells (ECs) were identified by
immunofluorescence staining with anti-vWF antibody followed by Alexa
488-conjugated secondary antibody (green channel). Nuclei were
counterstained with 4,6-diamidino-2-phenylindole (DAPI; blue
channel). Magnification is ×400 in all panels.
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Fig. 5.
Immunohistochemical detection of HSPG core
proteins in paraffin sections. A, normal brain
vessel stained with anti- HS antibody 3G10; B, high-grade
glioma vessel stained with anti-
HS antibody 3G10; C,
normal brain vessel stained for syndecan-4 (sdc4);
D, high-grade glioma vessel stained for syndecan-4;
E, normal brain vessel stained for glypican-1
(gpc1); F, high-grade glioma vessel stained for
glypican-1; G, high-grade glioma vessel stained with the
endothelial marker CD31; H, high-grade glioma vessel stained
for syndecan-1 (sdc1). The inset shows positive
control tissue (breast duct (Du) with adjacent plasma cells
(PC)). Sections shown in A and B were
treated with heparitinase prior to antibody incubation. Magnification
is ×400. EC, endothelial cells; TC, tumor
cells.
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Fig. 6.
Detection of HSPG core proteins in human
glioma and normal brain tissues by immunofluorescence. Frozen
sections from human high-grade gliomas (glioblastoma multiforme
(GBM)) and from normal brain controls
(nml) were stained with antibodies to syndecan-2 and
-3 and glypican-1 using Alexa 546-conjugated secondary antibody for
detection (red channel). An irrelevant mouse anti-hemagglutinin
antibody served as a negative control (NC). Blood vessels
were localized on the same sections using rabbit anti-vWF antiserum and
Alexa 488-conjugated secondary antibody for detection (green channel).
Panel pairs display the red channel as a gray-scale image to allow
better evaluation of staining intensity (A-D and
I-L) and the same images in dual-label mode (anti-core and
anti-vWF antibodies) for evaluation of co-localization (E-H
and M-P). Insets in E-H and
M-P show vascular endothelial cells at higher
magnification. A and E, glioma vessels stained
for syndecan-2. Please note antibody reactivity surrounding the blood
vessel (arrowhead in the inset in E),
but only weak-to-moderate staining in the vascular endothelial cells.
B and F, glioma vessels stained for syndecan-3.
Moderate staining was seen in endothelial cells. C and
G, glioma vessels stained for glypican-1. Strong and diffuse
staining was present in tumor vessel endothelial cells. D
and H, glioma vessels stained with irrelevant control
antibody. Some background staining was present in the tumor cells, but
not in the blood vessels. I and M, normal brain
vessels stained for syndecan-2. Only weak staining was present in
endothelial cells. J and N, normal brain vessels
stained for syndecan-3. Moderate-to-strong staining was present in
endothelial cells. K and O, normal brain vessels
stained for glypican-1. Only very weak staining was present in
endothelial cells. L and P, normal brain vessels
stained with irrelevant control antibody. Magnification is ×200 in the
main panels and approximately ×600 in the insets.
TV, tumor vessel; NV, normal vessel.
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Fig. 7.
Effect of glypican-1 and syndecan-1
overexpression on FGF2-induced proliferation of brain endothelial
cells. A-C, stable transfection of MBE cells with
glypican-1 (Gpc1). A, HSPGs extracted from MBE
cells stably transfected with mouse glypican-1 cDNA and from
mock-transfected cells were analyzed by SDS-PAGE. The membrane was
blotted with anti- HS antibody 3G10 to detect all HSPGs produced by
these cells. B, BrdUrd incorporation was determined as a
measure of proliferation. Base-line growth was measured in the absence
of FGF2. C, proliferation was measured in response to
increasing concentrations of FGF2. Response curves were normalized by
defining base-line growth without added FGF2 as 0% and maximal growth
at the highest FGF2 concentration (100 pM) as 100%. Each
data point represents the mean of triplicates. Error
bars indicate S.E. The asterisk indicates a significant
difference by the Wilcoxon rank sum test. D-F,
stable transfection of MBE cells with syndecan-1 (Sdc1).
D, HSPGs extracted from MBE cells stably transfected with
mouse syndecan-1 cDNA and from mock-transfected cells were analyzed
by SDS-PAGE. The membrane was blotted with anti-
HS antibody 3G10 to
detect all HSPGs produced by these cells. E, base-line
growth was recorded in the absence of FGF2 as described for
B. F, FGF2 dose response was measured as
described for C.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENT |
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We thank Jens Eickhoff for statistical analyses of experimental data.
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FOOTNOTES |
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* This work was supported by American Cancer Society Research Scholar Grant RSG-01-068-01 (to A. F.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Dept. of Obstetrics and Gynecology, University of
Kiel, Michaelisstr. 16, D-24105 Kiel, Germany.
§ To whom correspondence should be addressed: Dept. of Pathology and Laboratory Medicine, University of Wisconsin, Clinical Sciences Center K4/850, Madison, WI 53562-8550. Tel.: 608-265-9283; Fax: 608-265-6215; E-mail: afriedl@facstaff.wisc.edu.
Published, JBC Papers in Press, February 18, 2003, DOI 10.1074/jbc.M211259200
2 D. Qiao and A. Friedl, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: FGF2, fibroblast growth factor-2; FGFR, fibroblast growth factor receptor; HS, heparan sulfate; HSPG, heparan sulfate proteoglycan; vWF, von Willebrand factor; MBE, mouse brain endothelial; HMVEC, human microvascular endothelial cell; BrdUrd, bromodeoxyuridine; AP, alkaline phosphatase; VEGF, vascular endothelial growth factor.
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