From the Laboratoire de Recherche sur la Croissance
Cellulaire, la Réparation et la Régénération
Tissulaires (CRRET), Unité Propre de Recherche de l'Enseignement
Supérieur Associées an CNRS (UPRES-A) CNRS 7053, Université Paris XII-Val de Marne, avenue du
Général de Gaulle, 94010 Créteil, France and the
¶ School of Biological Sciences, University of Liverpool,
Liverpool L69 7ZB, United Kingdom
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ABSTRACT |
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Heparin affin regulatory peptide (HARP) is a
polypeptide belonging to a family of heparin binding
growth/differentiation factors. The high affinity of HARP for heparin
suggests that this secreted polypeptide should also bind to heparan
sulfate proteoglycans derived from cell surface and extracellular
matrix defined as extracellular compartments. Using Western blot
analysis, we detected HARP bound to heparan sulfate proteoglycans in
the extracellular compartments of MDA-MB 231 and MC 3T3-E1 as well as
NIH3T3 cells overexpressing HARP protein. Heparitinase treatment of BEL
cells inhibited HARP-induced cell proliferation, and the biological activity of HARP in this system was restored by the addition of heparin. We report that heparan sulfate, dermatan sulfate, and to a
lesser extent, chondroitin sulfate A, displaced HARP bound to the
extracellular compartment. Binding analyses with a biosensor showed
that HARP bound heparin with fast association and dissociation kinetics
(kass = 1.6 × 106
M Heparin affin regulatory peptide
(HARP)1 belongs to a growing
group of heparin binding extracellular regulatory molecules, and it has
mitogenic and neurite outgrowth activities. Independently purified from
perinatal rat brain as a polypeptide that induces neurite outgrowth of
embryonic neurons (1), this protein, also named pleiotrophin (2), has
been purified from uterus (3) and adult brain (4) as a growth factor
for fibroblastic and endothelial cells. Expression of HARP mRNA is
developmentally regulated, and major expression is found during the
perinatal growth period in rat brain (5, 6), pointing to the possible function of this molecule in the maturation of nerve cells. Moreover, HARP mRNA is found in several adult tissues, and HARP has been implicated in tumor growth (7).
HARP cDNA has been cloned and sequenced from several species. The
predicted amino acid sequence has been determined in humans, mice, and
rats (2, 6, 8, 9) and shows 98% homology among the three species. In
addition, the HARP amino acid sequence shares 55% homology with the
midkine gene product (10). In contrast to their neurite outgrowth
activity, initial studies differed in their results about the mitogenic
activity of midkine and HARP (3, 9, 11-15), possibly because of
differences in the cell type used or to the isolation procedure. More
recent studies clearly demonstrate that this family of polypeptides
have growth-stimulatory activity (16).
The high affinity of HARP for heparin suggests that HARP may bind to
heparan sulfate proteoglycans (HSPGs) present in extracellular compartments defined as cell surface and extracellular matrix (ECM).
Recent studies have demonstrated that syndecan-1, syndecan-3 (N-syndecan), and syndecan-4 (ryudocan) bind HARP with high
affinity (13, 17, 18). In addition, biochemical and cell biological studies have pointed to syndecan-3 as the HARP receptor involved in
neurite outgrowth activity (19). Despite the correlation derived from
previous studies, there has been no direct biochemical demonstration
that HARP is trapped in the extracellular compartment as a mitogenic
molecule bound to HSPGs. In this study we investigated whether HARP is
present in the extracellular compartment as well as the role of
glycosaminoglycans (GAGs) in controlling HARP mitogenic activity.
Materials--
Cell culture reagents were from Life
Technologies, Inc. Heparin lyase I (Flavobacterium heparinium; EC
4.2.2.7), heparin lyase III (F. heparinium; EC
4.2.2.8), chondroitin ABC lyase (Proteus vulgaris; EC
4.2.2.4), leupeptin, pepstatin, phenylmethylsulfonyl fluoride,
heparan sulfate (HS) from bovine intestinal mucosa, keratan sulfate
(KS) from bovine cornea, chondroitin sulfate A (CS-A) from bovine
trachea, dermatan sulfate (DS) from porcine skin, and chondroitin
sulfate C (CS-C) from shark cartilage were purchased from Sigma.
Horseradish peroxidase-conjugated goat anti-rabbit immunoglobulins were
obtained from Diagnostics-Pasteur (Marne la Coquette, France). The
affinity-purified HARP antibodies were obtained as described previously
by Ledoux (20). Heparin-Sepharose was from Amersham Pharmacia Biotech,
Immobilon P and ECL chemiluminescence were purchased from Millipore
Corporation (Saint Quentin en Yvelines, France), and Amersham Pharmacia
Biotech, respectively. Porcine mucosal heparin was a kind gift from M. Petitou (Sanofi, France), ABC alkaline phosphatase substrate kit 1 and
Levamisol were from Biosys, Vector laboratories (Compiégne,
France). FGF2 was a gift from G. Mazue (Amersham Pharmacia Biotech).
Cell Culture--
Bovine epithelial lens (BEL) cells were
isolated by the method previously described (21). The cells were grown
in DMEM supplemented with 10% fetal calf serum in the presence of 5 ng/ml FGF2 and were used between passages 10 and 20. NIH3T3 cell lines
stably transfected with pJK12 that overexpressed HARP (H-NIH3T3) were cultured as described previously (22). MDA-MB 231 cells were a gift
from M. Crépin (Bobigny, France), and MC 3T3-E1 cells were a gift
from M. Meunier (Lyon, France).
Western Blot Analysis of HARP--
The presence of HARP in
extracellular compartments defined as cell surface and extracellular
matrix was investigated by washing the cells grown to confluency in a
150-cm2 tissue culture dish with 2 × 5 ml of 20 mM Hepes, pH 7.4, containing 2 M NaCl
supplemented with 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, 1 µg/ml pepstatin A, and 5 mM EDTA.
The 2 M NaCl washes diluted 1:4 and the conditioned medium
of cells were incubated for 4 h with 100 µl of 10% (w/v)
heparin-Sepharose at 4 °C on a shaker. The heparin-Sepharose was
washed four times with 10 ml of 20 mM Hepes, pH 7.4, 0.5 M NaCl and twice with 10 ml of 20 mM Hepes, pH
7.4. Bound proteins were eluted with 50 µl of 2× Laemmli sample
buffer (23) under reducing conditions. Samples were boiled for 3 min,
and then polypeptides were separated on SDS-15% polyacrylamide gels.
Polypeptides were electrophoretically transferred to Immobilon P in 50 mM CAPS buffer, pH 11, containing 10% methanol for 20 min
at 200 V using a semi-dry apparatus Sartoblot II (Sartorius, Palaiseau,
France). Membranes saturated with 3% (w/v) gelatin in phosphate-buffer
saline (PBS) supplemented by 0.2% (v/v) Tween 20 were incubated with 1 µg/ml affinity-purified HARP antibody. Antibodies were detected by
using horseradish peroxidase-conjugated goat anti-rabbit IgG and ECL
chemiluminescence according to the manufacturer's recommendations.
Mitogenic Activity--
Growth-promoting activity was determined
by measuring [methyl-3H]thymidine incorporation into DNA
of BEL cells. Cells were seeded at 104/well in 48-well
culture plates in DMEM supplemented with 10% (v/v) fetal calf serum.
After 72 h, the medium was discarded, and the cells were
maintained for 24 h in serum-free DMEM. Aliquots of human
recombinant HARP were diluted in PBS containing 0.1% (w/v) bovine
serum albumin and added to triplicate wells for 18 h. Cells were
then pulsed with 0.5 µCi of [methyl-3H]thymidine/well
for 6 h. Radiolabeled DNA was precipitated with 250 µl of 10%
(w/v) trichloroacetic acid for 20 min at 4 °C, washed with tap
water, and solubilized with 250 µl of 0.1 M NaOH for 30 min at 37 °C. [Methyl-3H]thymidine incorporation was
determined by scintillation counting. The effect of GAGs on
[methyl-3H]thymidine incorporation induced by HARP was
investigated essentially as described previously (24). Assays were
performed at least in duplicate.
Release of Extracellular Compartment-bound HARP by Enzymatic
Treatment or Exogenous Addition of GAGs--
NIH3T3 cells transfected
with HARP cDNA were seeded at a density of 4 × 104/cm2 in 150-cm2 tissue culture
dishes. After 72 h, confluent cultures were washed twice with PBS
and incubated for 2 h at 4 °C in DMEM alone or in DMEM
containing 1.2 international milliunits/ml heparinase, heparitinase, or
chondroitinase ABC or various concentrations of GAGs ranging from 0.1 to 100 µg/ml. The medium was removed, and the cells were washed twice
with 20 mM Hepes, pH 7.4, containing 0.15 M
NaCl, 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml
leupeptin, 1 µg/ml pepstatin A, and 5 mM EDTA. HARP was
detected in the extracellular compartments of treated cells by Western
blot as described above. ECM was prepared from MC 3T3-E1 cells
essentially as described by Vlodavsky (25). Briefly, MC 3T3-E1 cells
were grown to confluency. The culture medium was removed, and the cells
were washed twice with PBS. The ECM was exposed by dissolving the cell
layer with 20 mM NH4OH containing 0.05% (v/v)
Triton, followed by five washes with PBS. It is noteworthy that using
this treatment, no residual cell surface material has been identified
on the insoluble matrix (7). HARP was extracted by scraping the ECM in
Hepes buffer containing 2 M NaCl. Dilutions (1:4) of the 2 M NaCl fractions were analyzed by Western blot as described above.
Immunohistochemistry--
MC 3T3-E1 cells were cultured on
chamber slides (LabTek, Nunc, Inc). ECM was prepared as described above
and fixed in acetone at 4 °C for 10 min. After three washes in PBS,
cells were incubated with 1.5% (v/v) normal goat serum in PBS for 30 min and 90 min at 37 °C with HARP antibodies (1 µg/ml)
diluted in PBS, 0.2% (v/v) Tween 20. Immunoreactivity was revealed by
using the ABC alkaline phosphatase substrate kit 1 supplemented with 1 mM Levamisol.
Activation of HARP by Heparin in Heparitinase-treated BEL
Cells--
BEL cells were seeded in 48-well plates in DMEM medium
supplemented with 10% (v/v) fetal calf serum as described above and incubated for 72 h. The medium was then discarded, and the cells were maintained in serum-free DMEM. Twenty h later the cells were treated with 3 international milliunits/ml heparitinase and incubated for 4 h at 37 °C. The medium was discarded, and the cells were incubated in serum-free DMEM in the presence of 1.25 ng/ml HARP (ED40: efficiency dose for 40% of maximal stimulation
induced by HARP) with or without increasing concentrations of heparin (10 to 104 ng/ml) in triplicate for 18 h, and the
incorporation of [methyl-3H]thymidine incorporation was
determined as described. Experiments were performed in duplicate.
Binding Assays--
Porcine intestinal mucosa heparin was
purified and biotinylated as described (26). DS was biotinylated in a
similar fashion. Five hundred µg of DS in 100 µl of distilled water
was incubated with 30 µl of a 50 mM solution of
N-hydroxysuccinimide amino caproate-LC biotin
(Pierce-Warriner, Chester, UK) in dimethyl sulfoxide for 72 h.
Unreacted biotin was removed by fractionation on a Sephadex G-25 column
(1 × 25 cm) equilibrated in distilled water, and DS was
lyophilized. Biotinylated DS was immobilized on
streptavidin-derivatized planar surfaces as described for biotinylated
heparin (26).
Binding reactions were carried out in an IAsys resonant mirror
biosensor at 20 °C using planar biotinylated surfaces derivatized with streptavidin according to the manufacturer's instructions (Affinity Sensors, Saxon Hill, Cambridge, UK). HARP binding assays were
repeated at least twice. The distribution of the immobilized heparin,
DS, and of the bound HARP on the surface of the biosensor cuvette was
inspected by examination of the resonance scan, which showed that at
all times these molecules were distributed uniformly on the sensor
surface and therefore were not microaggregated. Binding assays were as
described (27), and the amount of bound HARP is reported in arc s (1 arc s = 1/3600°. Briefly, the ligate, HARP, was added at a known
concentration in 100 µl of PBS-T (PBS supplemented with 0.02% (v/v)
Tween 20), and then the association reaction was followed over a set
time, usually 150 s. The cuvette was then washed twice with 200 µl of PBS-T, and the dissociation of bound ligate into the bulk PBS-T
was followed over time. To remove residual bound ligate and thus
regenerate the immobilized ligand, the cuvette was washed twice with
200 µl of 2 M NaCl, 10 mM
Na2HPO4, pH 7.2. Binding parameters were
calculated from the association and dissociation rate constants,
kass and kdiss, respectively, using the nonlinear curve-fitting FastFit software (Affinity Sensors) provided with the instrument, as described (27).
HARP did not itself bind to streptavidin-derivatized surfaces.
Presence of HARP in the Extracellular Compartments--
The
presence of HARP in the culture medium conditioned by MDA-MB 231 (7)
and MC 3T3-E1 (28) cells prompted us to determine whether HARP was also
present in the 2 M NaCl washes of the surface of these
cells, which corresponded to most of the material bound to the ECM and
cell surface. In MDA-MB 231 (Fig.
1A) and MC 3T3-E1 cells (Fig.
1B), HARP immunoreactivity was observed both in the conditioned medium (lanes 1) and in the 2 M NaCl
washes (lanes 2). Thus, HARP is secreted by cells into the
culture medium as well as sequestered in their extracellular
compartments.
To determine whether HARP was associated with the ECM, the latter was
prepared from MC 3T3-E1 cells that produced a substantial ECM and
tested for the presence of HARP by immunocytochemistry. Immunocytochemistry of ECM preparations displayed intense staining by
anti-HARP (Fig. 2A). In
contrast, no immunostaining was observed when nonspecific antibodies
were used (Fig. 2B) or after a 2 M NaCl wash
(result not shown). Furthermore, as shown in Fig. 2C, Western blot analysis of 2 M NaCl washes of ECM
preparations showed HARP immunoreactivity.
Effect of Heparinase, Heparitinase, and Chondroitinase ABC on HARP
Release--
The finding that HARP was bound to the extracellular
compartments was reminiscent of other heparin binding growth factors like FGF2 (25, 29, 30), vascular endothelium growth factor (31), and
exon 6 platelet-derived growth factor (32). The high affinity of HARP
for heparin is consistent with the possibility that this molecule binds
to HSPGs. This was investigated by studying the effect of heparinase,
heparitinase, and chondroitinase ABC on the release of HARP from
H-NIH3T3 cells. Enzyme concentrations optimal for degradation of the
relevant GAGs were determined by using 35S-metabolically
labeled GAGs derived from H-NIH3T3 (result not shown). Treatment with
1.2 international milliunit/ml heparinase or heparitinase induced the
release of HARP from the extracellular compartments of NIH3T3 cells as
compared with the buffer alone (Fig. 3).
In contrast, treatment with chondroitinase ABC did not cause the
release of HARP beyond that observed in buffer alone (Fig. 3).
Effect of Glycosaminoglycans on HARP Release--
To confirm that
HARP binds to extracellular compartments through interactions with
HSPGs, we determined whether the exogenous addition of HSPGs could
release HARP from cellular binding sites. Cell monolayers of H-NIH3T3
cells were incubated at 4 °C with various GAGs, including HS, KS,
heparin, CS-A, DS, or CS-C at concentrations ranging from 0.1 to 100 µg/ml. Incubation with HS or heparin at 10 µg/ml resulted in the
efficient release of HARP (Fig.
4A). The same result was
observed with 100 µg/ml heparin or HS (result not shown). Studies
performed with other GAGs indicated that treatment with DS at 10 µg/ml or 100 µg/ml (result not shown) released a similar amount of
HARP as HS (Fig. 4A). In contrast, CS-A (10 µg/ml)
released considerably less HARP and CS-C (10 µg/ml), even less (Fig.
4A). KS (10 µg/ml) was without effect. NaCl (2 M) was able to release residual HARP (Fig. 4B).
Quantitative studies using indirect enzyme-linked immunosorbent assay
revealed that DS released up to 84% bound HARP, and HS released 78%.
In contrast, treatment with 100 µg/ml CS-A resulted in 40%
displacement of bound HARP, and no significant effect was observed with
CS-C or KS used at the same concentration (data not shown).
Characterization of the HARP-GAGs Interaction--
The HARP-GAGs
interaction was characterized by fast association kinetics, as
exemplified by HARP binding to immobilized DS (Fig.
5A). The dissociation of HARP
from immobilized DS was also fast (Fig. 5B). The binding of
HARP to heparin and DS was always homogenous; there was no evidence for
the presence of more than one binding site for HARP in either of the
GAGs (Fig. 5B and Table I).
The association rate constant for the HARP-heparin interaction (kass = 1.6 (± 0.3) × 106
M
Competition binding assays were used to further characterize the
interactions between HARP and the GAGs. In the first set of
experiments, we examined whether HARP recognized a structural motif in
the GAGs that was also recognized by the archetypal heparin binding
growth factor, FGF2. The extent of binding of 111 nM HARP to DS was 97 ± 4 arc s (33, 34). The cuvette surface was then saturated with FGF2 by repeatedly adding 167 nM FGF2 until
no further binding was observed. The extent of binding of 111 nM HARP to the FGF2 saturated DS surface was similar to
that observed in the absence of FGF2, 92 ± 5 arc s. This result
suggests that HARP recognizes a structural motif distinct from that
recognized by FGF2. The same experiment was performed on the heparin
surface. The extent of binding of 55 nM HARP to the heparin
surface was 99 ± 3 arc s, whereas only 65 ± 10 arc s HARP
bound to the heparin surface saturated with FGF2, suggesting that the
structural motifs recognized by HARP and FGF2 in heparin may overlap to
a certain extent. In the second set of experiments, the ability of
different GAGs to inhibit the binding of 111 nM HARP to
immobilized heparin or DS was determined. On immobilized heparin (Fig.
5C), 140 arc s HARP was bound at equilibrium, and soluble
heparin was the most effective inhibitor of HARP binding, followed by
DS and CS-A. Thus 500 ng/ml heparin inhibited HARP binding by 87%,
whereas 50 µg/ml DS and 500 µg/ml CS-A reduced HARP binding by
79%. On immobilized DS (Fig. 5D), 53 ± 6 arc s HARP
was bound at equilibrium, and the rank order of the inhibitory ability
of the GAGs on HARP binding was the same as on immobilized heparin.
Thus 500 ng/ml heparin inhibited HARP binding by 82%, whereas 5 µg/ml DS was required for 85% inhibition, and 50 µg/ml CS-A for
88% inhibition.
Effect of Glycosaminoglycans on the Mitogenic Activity of
HARP--
We then examined whether HSPGs played a role in the
mitogenic response to HARP as demonstrated for the neurite outgrowth
activity by studying whether the response of heparitinase-treated BEL
cells to HARP was dependent on the presence of heparin. As shown in Fig. 6A, stimulation induced
by 1.25 ng/ml HARP was reduced by 50% in cells pretreated with
heparitinase. The addition of heparin in the concentration range from
10 to 104 ng/ml clearly restored and potentiated the
mitogenic activity of HARP. Maximum activity with heparin was seen at
100 ng/ml, corresponding to a stimulation of 4.2 times over the
control, i.e. cells treated with heparitinase and without
the addition of heparin. In contrast, no effect was observed when
heparin was added alone to untreated cells at concentrations ranging
from 10 to 104 ng/ml (result not shown). In our hands,
treatment of BEL cells with chondroitinase ABC resulted in the loss of
cell adhesion followed by cell death; these cells were thus unsuitable
for testing the role of chondroitin sulfate in the mitogenic response
(data not shown).
Since GAGs derived from the extracellular compartments are involved in
the binding of HARP, we investigated whether the addition of exogenous
GAGs modulated the mitogenic activity of HARP. As shown in Fig.
6B, a dual effect (potentiation/inhibition) was observed
when increasing concentrations of HS (10 ng/ml to 100 µg/ml) were
added, as described previously for heparin (24). Maximal potentiation
was observed at 1 µg/ml, whereas higher concentrations (10 to 100 µg/ml) inhibited the mitogenic activity of HARP. In control
experiments, HS used alone (10 ng/ml to 100 µg/ml) had no effect on
DNA synthesis. We also tested the effect of KS, CS-A, DS, and CS-C for
their ability to modulate the mitogenic activity of HARP. CS-A and DS
modulated the mitogenic activity of HARP. Potentiation occurred at
concentrations of CS-A and DS between 1 and 100 µg/ml; the maximum
effect was obtained at a concentration of 10 µg/ml, leading to a
2-fold increase in thymidine incorporation over that induced by HARP
alone. In contrast, KS (Fig. 6B) and CS-C (Fig.
6C) had no effect in the same range of concentrations. The
effect of DS on DNA synthesis induced by HARP or FGF2 on BEL cells was
also compared (Fig. 6D). At 1 µg/ml, HS leads to a 2- and
1.7-fold stimulation of DNA synthesis by FGF2 and HARP, respectively. As illustrated, CS-A and DS potentiate only the HARP stimulation, and
no effect of chondroitin sulfate was observed on FGF2 stimulation of
DNA synthesis in the range of concentrations tested (1-10
µg/ml).
Many growth factors bind to heparin, and the interaction of these
growth factors with HSPGs at the cell surface and in the extracellular
matrix is a central event in regulating the transport and effector
functions of the growth factors. The HS-FGF2 interaction is one of the
most extensively investigated and has at least two distinct functions.
The first function relates to HS acting as a local storage site for
FGF2. Several possible mechanisms has been demonstrated in the release
of biologically active FGF2 from HSPGs. FGF2 can be released from the
HS by an excess of soluble heparin or HS or by enzymatic digestion of
HSPGs. Enzymatic digestion by heparitinase (35, 36), plasmin (37), and
phospholipases (38, 39) generates soluble HS-FGF2 complexes that can
induce a mitogenic response. Secondly, FGF2 only stimulates a mitogenic response through its high affinity tyrosine kinase receptors if it also
interacts with HS (40). Other heparin binding growth factors like
transforming growth factor- Cells overexpressing HARP were used to study the specific properties of
HARP bound to the extracellular compartments. The presence of HARP in
the extracellular compartment was confirmed by Western blotting as an
18,000-Da polypeptide and by N-terminal sequencing (not shown).
Interestingly, 90% of the HARP from the extracellular compartment
preparation was inactive in our mitogenic assay. Sequence analysis of
this HARP isolated by Mono S chromatography showed that it had the
N-terminal sequence NH2-Gly-Lys-Lys-Glu-Lys-Pro. Comparison
with the N-terminal sequence of the mitogenic form supports the data
that the mitogenicity of HARP is directly or indirectly related to the
presence of three N-terminal amino acids (22).
Heparinase or heparitinase treatment of the cells, but not
chondroitinase treatment, released the extracellulary-stored HARP of
H-NIH3T3 cells, indicating that such HARP is associated almost exclusively with HS. In keeping with this conclusion, heparin and HS
were able to displace the extracellulary-stored HARP of H-NIH3T3 cells.
However, DS was also able to displace the extracellularly stored HARP
of H-NIH3T3 cells, although other GAGs that were tested were much less
effective. This result suggested that HARP may interact with DS,
although it is not associated with this molecule in the extracellular
compartment of H-NIH3T3 cells.
To further characterize the interactions between HARP and GAGs,
in vitro analyses were used to determine the binding
parameters of HARP for heparin and DS and to examine the relative
ranking of HARP-GAG interactions. The HARP-heparin interaction is
characterized by a fast association rate constant reminiscent of that
of hepatocyte growth factor/scatter factor for certain species of HS
(44) and by a fast dissociation rate constant. Consequently the
affinity of HARP for heparin is less than that of hepatocyte growth
factor/scatter factor for HS but higher than that of FGF2 for heparin
(26). The interaction of HARP with DS was characterized by a slower association rate constant and a slightly faster dissociation rate constant than observed with heparin; the affinity of HARP for DS
(Kd of 51 nM) is lower than that of the
hepatocyte growth factor/scatter factor-DS interaction, because the
association kinetics of the latter are considerably faster (45). HARP
bound to immobilized heparin, or DS was most efficiently displaced by heparin and then DS, in accordance with the relative affinities of
these GAGs for HARP. In contrast, CS-A was much less efficient (Fig.
5). These results support those obtained when different GAGs were used
to displace HARP from the extracellular compartment of H-NIH3T3 cells
(Fig. 4). Competition binding assays between HARP and FGF2 for binding
sites in DS and heparin reveal that, whereas these growth factors
recognize distinct binding sites in DS, in heparin, the binding sites
may overlap to a certain extent. The latter results may be explained by
structural studies that indicate that the 2-O-sulfated
iduronic acid units of heparin are important for the binding and the
neurite outgrowth activity of HARP, whereas the glucosamine
N-sulfate and 6-O-sulfate groups seem maybe less
important (18). In contrast, the 2-O-sulfated iduronic acid
and the N-sulfate groups of glucosamine play an important
role in FGF2 binding and biological activities (46).
There is good evidence that extracellular HS-like molecules are
required for the mitogenic signal induced by HARP. 1) Like heparin,
soluble HS potentiates DNA synthesis induced by HARP; 2) binding to
extracellular HS plays an important role in the growth-stimulatory
activity of HARP, because treatment with heparitinase
prevents this activity. However, the precise mechanism of action of
HARP is unknown. Although HSPGs molecules, including members of the
syndecan family, bind HARP with relatively high affinity, there is no
evidence that these cell surface proteins can act as receptors capable
of transducing the HARP mitogenic signal. Intriguingly, CS-A and DS
also potentiated HARP-induced stimulation of DNA synthesis in BEL
cells, and the equal potency of these GAGs (Fig. 6, C and
D) is in contrast to the greater affinity of HARP for DS
(Figs. 3 and 5).
It has also been reported that N-syndecan binds HARP with
high affinity (Kd = 600 pM) (13) and
plays an important role in the neurite outgrowth activity induced by
HARP. Indeed, heparitinase treatment of cells abolishes the neurite
outgrowth induced by HARP, clearly demonstrating the key role of
N-syndecan (19) or similar HSPGs in HARP-stimulated neurite
outgrowth. Small amounts of exogenous heparin and HS both inhibit
HARP-induced neurite outgrowth, with ID50 (dose that
induced 50% inhibition) values of 25 ng/ml and 700 ng/ml,
respectively (47). In this study we found that a low
concentration of exogenous GAGs potentiated rather than inhibited
the mitogenic effect of HARP, suggesting that HARP mitogenic and
neurite outgrowth activities might occur via independent mechanisms.
In addition to modulating the mitogenic activity of HARP, DS and HS are
likely to regulate the diffusion of HARP within and between cellular
compartments. Heparin and DS bind HARP with different affinities and
kinetics. Therefore, the relative distribution of HS and DS in
mesenchyme, basement membrane, and on epithelial cells at particular
stages of tissue development could either be permissive or
nonpermissive for the diffusion of HARP from the mesenchyme to the
epithelium. For example, the presence of a large number of the higher
affinity heparin/HS-type binding sites in the basement membrane coupled
to predominantly DS binding sites on target cells would result in an
accumulation of HARP in the basement membrane rather than its diffusion
to its target cells. Studies of the mechanism whereby DS and HS bind
HARP and modulate its activity represent an important challenge to the understanding of the biological function of this growth factor.
1 s
1;
kdiss = 0.02 s
1), yielding a
Kd value of 13 nM; the interaction
between HARP and dermatan sulfate was characterized by slower
association kinetics (kass = 0.68 × 106 M
1 s
1) and a
lower affinity (Kd = 51 nM). Exogenous
heparin, heparan sulfate, and dermatan sulfate potentiated the
growth-stimulatory activity of HARP, suggesting that corresponding
proteoglycans could be involved in the regulation of the mitogenic
activity of HARP.
INTRODUCTION
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ABSTRACT
INTRODUCTION
REFERENCES
EXPERIMENTAL PROCEDURES
RESULTS
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Fig. 1.
Presence of HARP in the extracellular
compartments of MDA-MB 231 and MC 3T3-E1 cells. Conditioned medium
(lane 1) or 1:4 dilution of 2 M NaCl washes of
the cell surface (lane 2) was incubated with 100 µl of a
10% (w/v) suspension of heparin-Sepharose. Bound protein was eluted
with Laemmli sample buffer and analyzed by Western blot as described in
under "Experimental Procedures." A, samples obtained
from MDA-MB 231 cells. Lane 1, conditioned medium;
lane 2, 2 M NaCl washes of the cell surface.
B, samples obtained from MC 3T3-E1 cells. Lane 1,
conditioned medium; lane 2, 2 M NaCl washes of
the cell surface.
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Fig. 2.
HARP immunoreactivity staining of isolated
ECM. MC 3T3-E1 cells were grown to confluence, and ECM was
prepared by NH4OH incubation as described under
"Experimental Procedures." Immunoreactivity was detected by using
affinity-purified anti-HARP immunoglobulins (1 µg/ml) (A)
or nonspecific immunoglobulin fractions (1 µg/ml) as negative control
(B). C, a 1:4 dilution of 2 M NaCl
wash of the extracellular matrix of MC 3T3-E1 was incubated with 100 µl of 10% (w/v) suspension of heparin-Sepharose. Bound proteins were
eluted with Laemmli sample buffer and were analyzed by Western blot as
described under "Experimental Procedures."
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Fig. 3.
Western blot analysis of the effect of
enzymatic treatment on HARP release from extracellular
compartments. H-NIH3T3 cells were seeded at 6 × 106 cells in 150-cm2 tissue culture dishes.
Conditioned medium was removed 72 h later. Cells were washed with
PBS and incubated for 2 h at 4 °C with DMEM (lane
2), DMEM containing 1.2 international milliunits/ml heparinase
(lane 3), DMEM containing 1.2 international milliunits/ml
heparitinase (lane 4), or DMEM containing 1.2 international
milliunits/ml chondroitinase ABC (lane 5). HARP was detected
in the incubation medium by Western blotting, as described under
"Experimental Procedures." Lane 1, 20 ng of purified
HARP.
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Fig. 4.
Effect of glycosaminoglycans on HARP
release. Cell monolayers of NIH3T3 cells were incubated with
various GAGs (10 µg/ml) including heparin (lane 2), HS
(lane 3), KS (lane 4), CS-A (lane 5),
DS (lane 6), CS-C (lane 7), or with incubation
buffer alone (lane 1). Cells were treated as described under
"Experimental Procedures," and the presence of HARP in GAG-treated
cells (A) or treated with GAGs as described in A
following treatment with 2 M NaCl washing buffer
(B) was performed by Western blot.
1 s
1) was twice as fast as
that for the HARP-DS interaction (kass = 0.68 (± 0.07) × 106 M
1
s
1) (Table I). The dissociation rate constant of HARP
from heparin was slightly slower than that of HARP from DS (Table I).
Thus, when the kinetic parameters were used to calculate the affinity of the HARP-GAG interactions, the interaction between HARP and heparin
had a considerably higher affinity (Kd 13 ± 3 nM) than the interaction between HARP and DS
(Kd 51 ± 14 nM). The
KD values calculated from the extent of binding observed at equilibrium were very similar to those calculated from the
kinetic binding parameters (Table I).
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Fig. 5.
Interaction of HARP with GAGs.
Biotinylated DS (A and D) or heparin
(C) was immobilized on a streptavidin-derivatized
aminosilane surface as described under "Experimental Procedures."
A, the binding of different concentrations of HARP (14-555
nM) to immobilized DS was followed in real time for 80-110
s, and then, after two quick washes with 200 µl of PBS-T, the
dissociation of the bound HARP into 200 µl of PBS-T was followed for
the next min. Two independent sets of binding reactions were performed,
of which one is presented. B, a plot of
kon against ligand concentration yields a
straight line (r = 0.973), the slope of which
corresponds to the association rate constant,
kass = 0.68 (± 0.07) × 106
M 1 s
1. The
kon of HARP for DS at each concentration of HARP
was determined using the FastFit software. Competition for HARP bound
to immobilized heparin (C) or DS (D) by various
GAGs. The binding of HARP (111 nM) was followed in the
biosensor in presence of various concentrations of GAGs, as indicated
in the figure.
, control;
, heparin;
, CS-A;
, CS-B.
Kinetics of HARP binding to immobilized heparin and DS
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Fig. 6.
Effects of GAGs on the mitogenic activity of
HARP. A, HARP activation assays by various
concentrations of heparin were carried out using BEL cells treated with
3 international milliunits/ml heparitinase as described under
"Experimental Procedures." [3H]thymidine
incorporation is expressed as compared with positive control values.
The bars indicate the S.E. of triplicate measurements.
B, concentration-response curves of DNA synthesis by cells
stimulated (filled symbols) or unstimulated (open
symbols) by HARP in the absence of serum and the presence of HS
( ,
) or KS (
,
). C, concentration-response
curves of DNA synthesis by cells stimulated (filled symbols)
or unstimulated (open symbols) by HARP in the absence of
serum and in the presence of CS-A (
,
), DS (
,
), or CS-C
(
,
). DNA synthesis was determined by measuring
[3H]thymidine uptake from 18 to 24 h after addition
of GAGs. D, potentialization by GAGs of DNA synthesis
stimulations induced by HARP and FGF2 in BEL cells. Stimulations
induced by HARP (filled bars) and FGF2 (open
bars) in the presence of various concentrations of HS, CS-A, and
DS were compared with the stimulations induced by the growth factors
alone and referred as 100% relative DNA synthesis. The bars
indicate the S.E. of triplicate measurements.
DISCUSSION
1 and -2 (41), exon 6 platelet-derived
growth factor (32), vascular endothelium growth factor (31), hepatocyte
growth factor/scatter factor (42), and heparin binding -EGF (43) are
associated with HSPGs present in the ECM, and the bioavailability and
activity of these growth factors is also regulated by HSPGs.
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ACKNOWLEDGEMENT |
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The authors thank G. Carpentier for his help in scanning illustrations.
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FOOTNOTES |
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* This work was supported by grants from Association pour la Recherche sur le Cancer (6595), Ministère de l'Education Nationale (DRED), and Ligue National contre le Cancer and Naturalia et Biologia.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.
§ Recipient of a grant from Association pour la Recherche sur les Tumeurs de Prostate.
Supported by the North West Cancer Research Foundation, the
Cancer and Polio Research Fund, and the Mizutani Foundation for Glycoscience.
** To whom correspondence should be addressed. Tel.: 33 1 45 17 17 97; Fax: 33 1 45 17 18 16; E-mail: courty{at}univ-paris12.fr.
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ABBREVIATIONS |
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The abbreviations used are: HARP, heparin affin regulatory peptide; HS, heparan sulfate; HSPG, HS proteoglycan; GAG, glycosaminoglycan; KS, keratan sulfate; CS-A, chondroitin sulfate A; CS-C, chondroitin sulfate C; DS, dermatan sulfate; FGF2, fibroblast growth factor 2 (basic fibroblast growth factor); BEL, bovine epithelial lens; DMEM, Dulbecco's modified Eagle's medium; ECM, extracellular matrix; PBS, phosphate-buffered saline; PBS-T, PBS with 0.02% (v/v) Tween 20; CAPS, 3-(cyclohexylamino)propanesulfonic acid.
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REFERENCES |
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