From the Department of Pathology, Anatomy, and Cell
Biology, the § Department of Medicine, Cardeza Foundation,
and the ¶ Cellular Biology and Signaling Program, Kimmel Cancer
Center, Thomas Jefferson University, Philadelphia, Pennsylvania
19107
Received for publication, October 11, 2002, and in revised form, November 14, 2002
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ABSTRACT |
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Perlecan, a ubiquitous basement membrane heparan
sulfate proteoglycan, plays key roles in blood vessel growth and
structural integrity. We discovered that the C terminus of
perlecan potently inhibited four aspects of angiogenesis: endothelial
cell migration, collagen-induced endothelial tube morphogenesis, and
blood vessel growth in the chorioallantoic membrane and in Matrigel
plug assays. The C terminus of perlecan was active at nanomolar
concentrations and blocked endothelial cell adhesion to fibronectin and
type I collagen, without directly binding to either protein; henceforth we have named it "endorepellin." We also found that endothelial cells possess a significant number of high affinity
(Kd of 11 nM) binding sites for
endorepellin and that endorepellin binds endostatin and counteracts its
anti-angiogenic effects. Thus, endorepellin represents a novel
anti-angiogenic product, which may retard tumor neovascularization and
hence tumor growth in vivo.
Perlecan is a modular proteoglycan that participates in the
formation and maintenance of basement membranes in various organs (1-5). The protein modules of perlecan have striking homology to
polypeptides involved in lipid uptake, growth control, cell-cell interactions, and adhesion (6-8). Its highly refined molecular architecture, coupled with its ubiquity, suggests that perlecan may
play key biological functions during ontogeny, tissue remodeling or
transformation (9, 10). Lack of perlecan causes embryonic lethality
with severe cephalic and cartilage abnormalities (11, 12). Although
basement membranes can develop in the absence of perlecan, the majority
of the perlecan-deficient mice succumb to intrapericardial hemorrhages
at day 10.5, when vasculogenesis is prominent and intraventricular
pressure rises (12). A recent report (13) has shown that perlecan-null
animals exhibit a high incidence of malformations of the cardiac
outflow tract with complete transposition of great vessels in ~73%
of the embryos, further stressing the key role of perlecan in
vasculogenesis. In adult tissues, perlecan is a major heparan sulfate
proteoglycan secreted by endothelial cells and is a potent inhibitor of
smooth muscle cell proliferation, a biological function mediated by
perlecan's block of FGF21
activity (14) and Oct-1 gene expression (15). Indeed,
perlecan is a major candidate for the FGF2 low affinity receptor and
can restore high affinity binding of FGF2 to its receptor in heparan sulfate-deficient cells (16). FGF2 binds to the heparan sulfate chains
attached to the N-terminal domain I, and the bioavailability of this
powerful angiogenic factor is modulated by the concerted action of
heparanases and proteases (17). In agreement with these data, perlecan
plays a critical role in regulating the vascular response to injury
in vivo (18).
Perlecan is highly enriched in various tumorigenic cell lines (19, 20)
and human tumors (21, 22), and blocking the endogenous production of
perlecan suppresses autocrine and paracrine functions of FGF2 and
impairs tumor cell growth and invasion (23, 24). Likewise, antisense
targeting of the perlecan gene causes a marked attenuation of colon
carcinoma cell growth, and these effects correlate with a reduced
mitogenic response to FGF7 (25). Perlecan protein core binds to FGF7
(Kd ~60 nM) (26), and is required for
functional activation of the FGF7 receptor and downstream signaling
(27). In addition, the perlecan protein core binds several
extracellular matrix molecules and growth factors (28) and acts as
either an adhesive or counter-adhesive molecule (29-32).
Angiogenesis is one of the most important events in tumor progression
and is greatly influenced by cell matrix interactions taking place at
the surface of the endothelial cells and the tumor-matrix boundaries
(33). Heparan sulfate proteoglycans act as depots for pro- and
anti-angiogenic factors (34, 35) and, in concert with members of the
FGF and VEGF family and their receptors, modulate various steps of
angiogenesis (10). Because expression of the full-length perlecan is
very difficult since its mRNA is over 15 kb, we utilized its C-terminal
domain V to search for physiologically relevant binding partners.
Domain V is the major cell-binding domain and is potentially a very
interactive molecule since it is organized in a structure similar to
that of agrin and various types of laminin, and it also binds heparin
and Cells, Yeast Two-hybrid Screening, and
Co-immunoprecipitation--
Primary cultures of HUVECs were prepared
from fresh umbilical cords and cultured on gelatin-coated flasks in
M199 or M200 media (Invitrogen) supplemented with 10% fetal bovine
serum, 50 µg/ml heparin, and endothelial cell growth supplement,
isolated from bovine hypothalami. Only passages 4-8 were used. A431
squamous carcinoma, HeLa squamous carcinoma, HT1080 fibrosarcoma, WiDr colon carcinoma, MCF7 breast carcinoma, and M2 mouse melanoma cells
were obtained from American Type Culture Collection (Manassas, VA). We
employed the Matchmaker GAL4 two-hybrid system 3 (Clontech, Palo Alto, CA), which adopts three
independent reporter genes (His, Ade, and either Expression and Purification of Recombinant Proteins--
The
pCEP-Pu vector bearing the sequence of the BM40 signal peptide and the
full-length domain V/endorepellin was electroporated into 0.5 ~106 human embryonic kidney cells (293-EBNA) expressing
the Epstein-Barr virus nuclear antigen (EBNA)-1. The endostatin
fragment was cloned by PCR using clone A3 as a template. The following
oligonucleotides were used: forward, 5'-CTAGCTAGCCCACAGCCACCGCGACTT-3';
containing an NheI site and reverse,
5'-CCGCTCGAGTACTTGGAGGCAGTCATGA-3' containing an XhoI site.
The GC-Rich PCR system was used (Roche Diagnostics). Mass cultures were
selected in media containing 250 µg ml Endothelial Cell Migration, Tube Formation, Chorioallantoic
Membrane (CAM), and Matrigel Plug Assays--
A 48-well Boyden chamber
(Neuroprobe Inc., Gaithersburg, MD) was used for HUVEC migration assays
with VEGF165 (R&D Systems, Minneapolis, MN) as a
chemoattractant. HUVECs migrated through 8-µm nucleopore,
polyvinylpyrrolidine-free polycarbonate filters (Corning, Cambridge,
MA) precoated for 48 h with 100 µg ml Binding Studies, Covalent Affinity Cross-linking, and Cell
Adhesion Assays--
Endorepellin (10 µg) was labeled to high
specific activity (~1018 cpm mol Expression of Endorepellin/Alkaline Phosphatase
Chimeric Protein and Binding Studies--
The heat-stable human
placental alkaline phosphatase (AP) was amplified by PCR from our
previously described construct (42) and ligated in-frame onto the C
terminus of endorepellin in the pCEP-Pu vector. The construct was
electroporated into 293-EBNA cells as described above. Following
several weeks in selective medium (250 µg ml Endostatin Is a Novel Interacting Partner for Perlecan Domain
V/Endorepellin--
To discover novel interacting partners
for perlecan protein core we utilized the entire domain V of perlecan
(7, 8), which we named endorepellin (amino acids 3687-4391, Fig.
1a) as bait and screened a
keratinocyte cDNA library in the yeast two-hybrid system. This
domain consists of three laminin-type G (LG1-LG3) modules separated by
four EGF-like (EG1-EG4) modules, in an arrangement highly conserved
across species (2, 43). One of the strongest interacting clones (clone
A3) encoded the C-terminal half of collagen type XVIII, including the
NC1 domain containing the potent anti-angiogenic factor endostatin
(Fig. 1b). Because endostatin inhibits endothelial cell
proliferation and effectively arrests the growth of several tumors
(44), and because perlecan and endostatin co-localize in most basement
membranes (2, 3, 45, 46), we reasoned that an interaction between these
two proteins could occur in vivo and could play a role in
tumor progression. Therefore, we subcloned the collagen fragment into
the pGADT7 vector, and the interaction with endorepellin was once more
tested with the yeast two-hybrid system on a one-to-one basis. The
growth of the cells in quadruple minus medium was comparable to that of
the positive control (pGBKT7-53/pGADT7-T), as well as the blue color
generated by
To establish a direct interaction between endorepellin and endostatin,
we performed several solid-phase binding assays using 125I-endorepellin as the soluble ligand, and endostatin,
fibronectin or collagen I as the solid substrates coated onto
Immulon wells. In these experiments, recombinant endorepellin and
endostatin were generated in 293-EBNA cells (see below), and the former
was radioiodinated to reach specific activities of ~1018
cpm mol Endostatin Interacts Specifically with the LG2 Module of
Endorepellin--
To establish the precise location of this
interaction, we generated seven deletion mutants ( Recombinant Endorepellin Is Anti-angiogenic--
Human recombinant
endorepellin, generated in 293-EBNA cells, migrated on SDS-PAGE as a
single band of the predicted ~81 kDa, whose identity was further
confirmed by immunoblotting with anti-His6 antibody (Fig.
3a) and ELISA using a specific
monoclonal antibody against domain V (17) (not shown). Because murine
perlecan domain V can be substituted with glycosaminoglycan side chains
(37, 48), we subjected 10 µg of purified endorepellin to DEAE
Sephacel chromatography. None of the purified endorepellin bound to the DEAE column under relatively low salt (NaCl, 150 mM)
concentrations (Fig. 3b, lane 3) indicating that
the human preparation did not contain glycosaminoglycan side chains.
Interestingly, our construct behaves like the Drosophila
perlecan domain V, which when expressed in the same 293-EBNA cells,
migrates as a single band without any overt glycanation (49).
To test the biological properties of endorepellin, we utilized
VEGF-induced migration of HUVEC to passages 4-8 (50). It is well
established that the motility and vectorial migration of endothelial
cells that coincidentally occur with invasion, are fundamental
components of angiogenesis (33, 51, 52). When VEGF was used in the
lower chamber, there was a complete suppression of HUVEC migration
through the membrane at 1-10 µg ml
Next, we investigated whether the inhibition of HUVEC migration could
lead to a decreased angiogenesis in vivo. Using the chorioallantoic membrane (CAM) assay, we discovered that endorepellin significantly reduced the angiogenic activity of VEGF (Fig.
4). In the presence of VEGF, the
characteristic spoke wheel-like vessel formation was induced toward the
sponge (Fig. 4a). In the presence of endorepellin (Fig.
4b), the vessel sprouts were markedly reduced to a level
comparable to the negative control (Fig. 4c).
Next, we tested whether endorepellin could counteract the angiogenic
stimuli of WiDr, a highly tumorigenic colon carcinoma cell line (53).
Indeed, long term culture of capillary endothelial cells was originally
obtained by culturing endothelial cells with media conditioned by
malignant cells (54). This indicates that tumor cells express a large
repertoire of growth-promoting factors that support endothelial cell
survival and proliferation. We observed that the presence of
endorepellin in the sponges harboring the colon carcinoma cells caused
a marked suppression of the angiogenic process (Fig. 4e) as
compared with the tumor cells themselves (Fig. 4d).
Quantification of both sets of experiments using the NIH Image analysis
software showed a 74 and 80% inhibition of vessel area around the
sponges (p < 0.001) (Fig. 4, f and
g, respectively).
To further investigate the role of endorepellin in in vivo
angiogenesis, we performed Matrigel plug assays in
nu/nu animals (40). To this end, we injected
~100 µl of Matrigel supplemented with FGF2 (10 ng/animal) and
either BSA or endorepellin (12 µg/animal) into the dorsal
subcutaneous regions of ten nu/nu mice. Mice were sacrificed 2 weeks
after the injection and the skin removed to analyze the blood vessel
formation. Inasmuch as the Matrigel plug is initially avascular, any
vessels found within the plug must be, of necessity, new vessels (40).
There were striking differences between control and
endorepellin-treated samples. In the latter case, there was a marked
inhibition of neovascularization around and within the Matrigel plug
(Fig. 5b) as compared with the
control samples (Fig. 5a). Microscopic examination showed
marked ingrowths of new blood vessels in the control samples (Fig.
5c), but essentially little or no blood vessel formation in
the presence of endorepellin (Fig. 5d). Quantification of
the blood vessel density, as described above, again showed a marked
(>75%) suppression of new blood vessels in the presence of
endorepellin.
Finally, we tested endorepellin during HUVEC tube formation in a
collagen matrix, a process thought to mimic morphogenesis (39). The
results showed a capillary-like network formation in the control HUVECs
(Fig. 5e), which was visible at 4 h, and remained
constant for up to 24 h (not shown). In contrast, endorepellin caused a complete block of tube-like formation at concentrations similar to those used in the migration assays (Fig. 5f),
whereas no significant effects were obtained with endostatin (Fig.
5g). Interestingly, endostatin was not capable of blocking
the activity of endorepellin (Fig. 5h), suggesting that
these two proteins possess distinct mechanisms of action (see below).
Collectively, our results indicate that endorepellin is a powerful
blocker of angiogenesis and that its effects are long lasting.
Biological Effects of Endostatin/Endorepellin
Interaction--
To further investigate the biological significance
of the interaction between endostatin and endorepellin, we performed
several VEGF-induced HUVEC migration experiments in which the amount of endorepellin was kept constant while the amount of endostatin was
proportionally increased. We chose two concentrations of endorepellin, 1.2 and 3.7 nM (100 and 300 ng ml Specific Binding of Endorepellin to Endothelial Cell
Surface--
Next, we sought to determine whether endorepellin could
specifically bind to the cell surface of HUVECs. We labeled
endorepellin with 125I to high specific activity
(~1018 cpm mol
The binding of endorepellin was saturable in the range of 10-20
nM (Fig. 7c). Scatchard analysis (Fig.
7d) revealed a single receptor population consisting of
~3.6 × 105 sites cell High Affinity Binding Sites for Endorepellin on Various Tumor Cell
Lines--
Next, we wished to test whether endorepellin-binding sites
could be present on cells other than HUVECs. To this end, we fused the
coding region of endorepellin to that of the heat-stable human placental AP (55), a soluble marker that can be readily detected by
chemiluminescence's reagents (42). We isolated several clones that
expressed relatively high levels of endorepellin/AP chimeric protein
(Fig. 8a) and quantitative
analysis, using a standard curve based on AP activity, revealed that
105 clone 6 cells expressed ~4 µg ml Endorepellin Has Counter-adhesive Properties for Endothelial
Cells--
A number of bioactive fragments of extracellular matrix
proteins exhibit counter-adhesive activity; that is, they disrupt cell-matrix interactions (52). It has been previously shown that domain
V of perlecan, from either mouse or Drosophila, is adhesive
for several cell lines when compared with fibronectin, but not for
others (36, 49). To address this point, we tested whether endorepellin
could mediate HUVEC adhesion. We found a complete lack of HUVEC
adhesion to either endorepellin or BSA, in contrast to a robust
adhesion to fibronectin or collagen type I (Table
I). In competition experiments in which
we challenged HUVECs with increasing amounts of endorepellin, we found
a progressive inhibition of HUVEC attachment; within minutes the cells
rounded up and began to detach in a dose-dependent manner
(Fig. 9a). We performed
several experiments on fibrillar collagen or plastic and, consistently,
endorepellin prevented HUVEC binding to either substratum, with an
IC50 of 5-20 nM. In contrast, endostatin did not show any interference with endothelial cell attachment to either
fibronectin or collagen I (data not shown).
To verify that the anti-adhesive property of endorepellin was not just
limited to endothelial cells, we tested HT1080 fibrosarcoma cells,
which do not bind to murine domain V (36), and WiDr colon carcinoma
cells (19). In both cases, endorepellin did not support adhesion (Table
I). Moreover, specificity of endorepellin counter-adhesive properties
was confirmed by the efficient displacement of HT1080 and WiDr
attachment to fibronectin with increasing concentrations of recombinant
endorepellin, with IC50 of 110 and 40 nM,
respectively (Fig. 9b). In contrast, endostatin did not
significantly affect the adhesion of either cell line (Fig.
9c). A summary of all the binding data, in which adhesion
assays were performed using fibronectin and two concentrations of
collagen type I, is provided in Table I. Interestingly, we found that
endorepellin not only failed to support adhesion for HUVECs, but also
for most of the tumor cell lines tested, including HeLa, HT1080, WiDr,
and M2 tumor cell lines. In contrast, A431 squamous carcinoma cells,
which were previously shown to adhere to murine domain V, showed a mean attachment value of 52 ± 4% nearly identical to what has been previously obtained (36). We also found that MCF7 breast carcinoma cells had a similar (50 ± 7%) attachment value. Thus,
endorepellin is a powerful anti-adhesive factor for endothelial cells
and certain tumor cells, while it is partially adhesive for other tumor
cell lines.
In an in vivo screening using the entire C-terminal
domain V of human perlecan as bait, we discovered a strong interacting protein comprising the C terminus of human collagen type XVIII, including the anti-angiogenic factor endostatin (44). It has been
previously shown, using a cell-free system, that perlecan proteoglycan
binds to endostatin, presumably via the heparan sulfate chains (56,
57). We independently confirm these results and further show that a
distinct subdomain of perlecan protein core specifically binds to
endostatin. Using a battery of deletion mutants, the major binding site
was mapped to the second laminin-like G domain of perlecan domain V. Because perlecan and type XVIII collagen/endostatin co-distribute in
basement membranes (3, 22, 45, 46, 56), and because endostatin binds
in situ to vascular basement membranes independently of
heparan sulfate (58), we propose that domain V is a binding site for
endostatin in vivo. Therefore, one outcome of these results,
from a physiological point of view, would be that we have discovered an
important interaction between the C terminus of perlecan and the C
terminus of type XVIII collagen. This interaction could play a key role
in the assembly of basement membranes and, perhaps, in the maintenance of their integrity.
Surprisingly, using HUVEC migration assays, we discovered that, while
the interaction between endostatin and domain V counteracted their
activities, perlecan domain V itself was a powerful anti-angiogenic factor, and hence we named it endorepellin. Endorepellin was active at
nanomolar concentrations and was a potent inhibitor of angiogenesis in
four independent assays commonly used to study angiogenesis: endothelial cell migration through fibrillar collagen, collagen-induced capillary-like formation, and growth of blood vessels in the CAM and
Matrigel plug assays. The action of endorepellin was as strong as
endostatin in inhibiting HUVEC migration, and in some experiments was
even stronger than endostatin. Interestingly, endorepellin was also
capable of counteracting the angiogenic properties of WiDr colon
carcinoma cells in the CAM assays. Notably, these cells synthesize
large amounts of perlecan (19), which has been recently shown to bind
FGF2 with affinities even higher than the endothelial cell perlecan
(59). Thus, it is possible that endorepellin might act in a negative
dominant fashion, at least in regard to the inhibition of capillary
formation. We recently found that 293-EBNA cells expressing
endorepellin do not form tumors in nude mice, in contrast to the
wild-type cells,2 suggesting
that endorepellin might also play an anti-tumorigenic role in
vivo.
We found a significant number (~3.6 × 105
cell Endorepellin interfered with the adhesive properties of endothelial
cells for various substrata, including fibronectin and fibrillar
collagen, without directly binding to either protein matrix, and was
also anti-adhesive for certain tumor cells derived from colon,
neuroectoderm or mesenchyme. This is in agreement with previous studies
showing anti-adhesive properties for perlecan in hematopoietic (30),
mesangial (31), myoblastic (60), and smooth muscle (61) cells, and a
role for perlecan in the suppression of growth and invasion in
fibrosarcoma cells (62). However, while endorepellin inhibits tube
formation and prevents cell adhesion to fibronectin and other
substrata, monomeric endostatin does not. Thus, the two molecules may
act via distinct mechanisms. We should point out, however, that
oligomeric endostatin and the NC1 domain of collagen type XVIII have
been recently shown to effectively inhibit tube morphogenesis (63),
indicating that oligomerization is an important feature for their
activity. In support of this concept, deletion of cle-1 NC1
domain, the Caenorhabditis elegans homologue of mammalian
collagen XVIII, causes defects in cell migration and axonal guidance
(64). Both of these defects can be rescued by ectopic expression of the
NC1 domain, known to trimerize in vitro, but not by the
monomeric endostatin domain.
Notably, the potent inhibitory activity on endothelial cell migration
by endorepellin was neutralized by endostatin. Presumably, this occurs
by the tight binding of endostatin to endorepellin that would alter the
ability of endorepellin to interact with the cell surface. A logical
extension of this hypothesis would be that this binding would also
block the other activity (tube formation) of endorepellin. However, we
observed that endostatin did not neutralize this activity. Migration of
endothelial cells and tube-like formation are two different mechanisms
that involve activation of different pathways (33, 65). The former
occurs immediately after an angiogenic stimulus has taken place,
whereas the latter involves the differentiation of the endothelial
cells at the end of the angiogenic response. Thus, it is possible that the two proteins act on different receptors and that they activate similar or overlapping pathways during cell migration, but differ in
the morphogenetic process of tube-like formation within a collagen matrix. It is also possible that endorepellin may bind to more than one
receptor, each one involved in controlling different cellular mechanisms.
Powerful angiogenesis inhibitors are proteolytically processed forms of
basement membrane collagens types IV, XV, and XVIII, the latter two
being chondroitin and heparan sulfate proteoglycans, respectively (66).
Moreover, proteolytic remodeling of the extracellular matrix can expose
cryptic sites within collagen type IV that are required for
angiogenesis in vivo (67), Thus, it is likely that perlecan
might undergo a similar proteolytic processing in vivo, thereby liberating endorepellin through an endogenous processing mechanism common to most LG domains of laminin (43, 68, 69). The
modular nature of perlecan protein core is particularly well suited for
selective proteolysis (17, 66) and subsequent release of peptides with
biological activity. There are several lines of evidence that support
this scenario. First, in our 293-EBNA cells we detected a natural
25-kDa proteolytic cleavage product of endorepellin, which bound to the
Ni-NTA column and was also reactive with the anti-His6
antibody, indicating that it represented LG3. This was further
confirmed by N-terminal sequencing analysis, which perfectly matched
the seven amino acid residues starting with Asp-4197. Second, a similar
band was previously shown to represent a proteolytic fragment of murine
domain V generated by cleavage just before the beginning of LG3 (36,
37). This protease-sensitive region, which starts with the sequence
DAPGQYG, is completely conserved between mouse (6) and human (7, 8),
thus demonstrating that a specific cleavage of an Asn-Asp bond (at
positions 3514-3515 and 4196-4197, for the mouse and human
counterpart, respectively) had occurred near the N terminus of LG3.
Mutational analysis indicated that Asp, but not Asn, is crucial for
processing of mouse endorepellin (37), possibly by a specific, yet to
be discovered, Asp-N endoproteinase. In our study the LG3 module failed
to be cross-linked to surface proteins, suggesting that this part of
endorepellin is dispensable for surface binding. Third, an identical
proteolytic fragment of ~25 kDa, cleaved at the same position as the
mouse, was detected in the urine of patients with end-stage renal
failure (70). This indicates that the LG3 module is present in the
human serum at relatively high concentrations, since this LG3 was found
at concentrations of ~10 mg liter We do not yet know the precise mechanism of action of endorepellin. Two
cell-surface proteins might be involved, either separately or in
conjunction, namely, Recent experimental tests on tumor-bearing animals are encouraging
because protein-based inhibitors, such as endostatin, have the
following three major advantages. 1) They can reduce the tumors to a
manageable size. 2) They do not induce resistance, and 3) their
toxicity is low (66). Endorepellin is a novel natural inhibitor of
angiogeneis, and its use in cancer therapy has additional advantages
insofar as endorepellin may also exert an anti-adhesive action on
certain tumor cells. Thus, we predict that if these protein-based
agents are used in concert with traditional therapies, which target
neoplastic cells directly, we may manage, or even cure, some forms of
cancers that are currently incurable.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-dystroglycan among other molecules (36, 37). Thus, we employed
this fragment to screen a cDNA library in order to identify other
putative interacting proteins and to further investigate the function
and importance of this protein in the cross-talk between extracellular
matrix proteins. Using the yeast two-hybrid system, we identified
collagen XVIII, including the anti-angiogenic factor endostatin, as a
strong candidate. We discovered that the C terminus of perlecan,
henceforth named "endorepellin," counteracted the anti-angiogenic
effects of endostatin, while by itself potently inhibited four aspects of angiogenesis: endothelial cell migration, collagen-induced endothelial tube morphogenesis, and blood vessel growth in the chorioallantoic membrane and in Matrigel plug assays. Endorepellin inhibited angiogenesis at nanomolar concentrations and
interfered with endothelial cell adhesive properties for various
substrata, including fibronectin and type I collagen. Moreover,
endothelial cells possess a significant number of high affinity
(Kd of 11 nM) binding sites for
endorepellin, which could be cross-linked with
bis-sulfosuccimidyl-suberate (BS3) to form high
Mr complexes. Thus, endorepellin may represent a
novel anti-angiogenic tool against cancer.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
- or
-galactosidase) for selection. Endorepellin, subcloned into the
pGBKT7 vector, was used as bait to screen ~5 × 106
cDNAs from a keratinocyte library constructed in the pACT2 vector. The clones growing in selective medium were replated in quadruple minus
plates containing X-
-gal. Interacting cDNAs were identified by
automatic sequencing. Deletion mutants were generated by PCR using
oligonucleotides, which included suitable restriction sites to allow
unidirectional ligation into the pGBKT7 vector (38). Various constructs
were in vitro transcribed and translated in the presence of
[35S]methionine (ICN Pharmaceuticals, Costa Mesa, CA)
employing the TNT® reticulocyte lysate system (Promega,
Madison, WI). Aliquots were co-precipitated with affinity-purified,
anti-hemagglutinin (
HA) rabbit polyclonal antibodies
(Clontech). The immune complexes were captured with
protein A/G-agarose beads (Pierce), washed with 10 mM Hepes
pH 7.4, 150 mM NaCl, 0.1% Triton X-100, 10% glycerol, 200 mM Na3VO4, 20 mM NaF,
and a protease inhibitor mixture (Roche Diagnostics GmbH, Mannheim,
Germany), and separated in polyacrylamide gels. The gels were fixed in
ethanol/acidic acid, incubated for 20 min with AMPLIFYTM
(Amersham Biosciences), dried under vacuum, and exposed to Kodak films.
1 G418 and 500 ng
ml
1 puromycin. Serum-free conditioned media were
concentrated in a dialysis bag with polyethylene glycol, dialyzed, and
purified on Ni-NTA resin eluted with 250 mM imidazole. In
all the purification steps we used phenylmethylsulfonyl fluoride (2 mM) and N-ethylmaleimide (2 mM) as
protease inhibitors. Using this procedure, we routinely purified 5-10
mg of endorepellin or endostatin liter
1 of conditioned
medium. ELISA and immunoblotting with anti-domain V (17) or penta-His
(Qiagen, Valenica, CA) monoclonal antibodies were performed as
described before (26). About 15 pmol of purified LG3 fragment,
following separation in 10% SDS-PAGE and electroblotting onto a
Problott polyvinylidene difluoride membrane, were microsequenced using
Edman degradation (Applied Biosystems, Model 477A) at the protein
chemistry facility of the Kimmel Cancer Center. Purified perlecan
domain V (~10 µg) was diluted in 50 mM Tris-HCl,
containing 150 mM NaCl and further purified through a DEAE
Sephacel pre-equilibrated in the same buffer. Following an extensive
wash, the material was eluted with 2 M NaCl. Aliquots of
each fraction were analyzed by SDS-PAGE.
1 collagen
type I (Collaborative Biomedical Products, Bedford, MA). About 2 × 104 HUVECs were preincubated for 1 h with different
concentrations of endorepellin and/or endostatin from Pichia
pastoris (Calbiochem-Novabiochem, San Diego, CA), and allowed to
migrate through the filter for 6 h at 37 °C in 5%
CO2, with or without VEGF165 (10 ng
ml
1) in the lower chambers. The filters were then washed,
fixed, stained with Diff-Quick stain (VWR Scientific Products,
Bridgeport, NJ), and the transmigrated cells were counted using
conventional microscopy. For in vitro tube-like formation,
~4 × 105 HUVECs were seeded for 18 h onto
12-well dishes coated with 100 µg ml
1 collagen type I
and then covered with a second layer of collagen gel (39). Cultures
were incubated until gels had solidified, typically 15-30 min, and
then given 1 ml of serum-free media with the various test agents and
control substances. For the CAM assays, fertilized White Leghorn chick
eggs were incubated at 37 °C. After 3 days of incubation, ~3 ml of
albumin were removed to detach the CAM, and a small square window was
formed. The window was then sealed with tape, and the eggs were
returned to the incubator. At day 9, a 1-mm3 Gelfoam
sterile sponge (Gelfoam, Upjohn Company, Kalamazoo, MI) was placed on
the CAMs, and various test factors were applied. In addition, CAM
assays were performed using sponges containing either 0.5 × 106 WiDr colon carcinoma cells alone or in combination with
3 µg of recombinant endorepellin. Three-day-old embryos
(n = 20) were used in each experiment. The mean vessel
area was calculated using the NIH Image software program (version 1.61)
using at least four embryos per experimental point. Ten squares of
~500 µm2 each were randomly selected around the sponge
area and digitized. The background was uniformly adjusted so that it
would appear white, whereas the vessels would be black. The pixel
density of the vascularized areas was measured, and the values were
finally converted into surface area (µm2). Student's
two-sided t test was used to compare the values of the
experimental and control samples. A value of p < 0.05 was considered as statistically significant. Matrigel plug assays were
essentially performed as previously described (40). Briefly, 100 µl
of Matrigel (BD PharMingen, San Diego, CA) containing FGF2 (10 ng/animal), in the presence or absence of endorepellin (12 µg/animal), were injected into the dorsal subcutaneous regions of ten
nu/nu mice. Mice were sacrificed 2 weeks after the injection, and the
skin was removed to analyze the blood vessel formation. The skin
samples were photographed, fixed in buffered formaldehyde, and
processed for light microscopy. The newly formed blood vessels present
in the Matrigel plug were counted as detailed above.
1) using
Iodogen-coated tubes (Pierce). For saturation binding and Scatchard
analysis, confluent HUVECs in 24-well plates were incubated with
increasing concentrations of 125I-endorepellin for 2.5 h at 4 °C in M199 containing 0.1% BSA, washed several times, and
extracted in the presence of protease inhibitors (41). Estimates of
receptor affinity and total binding capacity were made with Sigma Plot
5.0 software. For covalent affinity cross-linking, HUVECs were
incubated with various concentrations of 125I-endorepellin
for 2 h, and then incubated for 30 min at 4 °C with 20 mM BS3, a membrane-impermeable cross-linker.
After termination of the reaction with 1 M Tris, pH 7.5, the cells were solubilized as described above, and the cross-linked
material was separated on SDS-PAGE and visualized by autoradiography
(27). Displacement of HUVEC-bound 125I-endorepellin was
performed by incubating confluent HUVECs (~105 cells per
dish) with 125I-endorepellin (5 nM) plus
increasing concentrations of recombinant unlabeled endorepellin. The
cells were incubated at 4 °C for 3 h, washed three times,
extracted, and counted in total as above. HUVECs and various tumor cell
lines were tested for adhesion to various substrata using various
coating concentrations (10-180 nM) of fibronectin,
collagen type I, BSA, endorepellin or endostatin as plastic-immobilized
substrata. The adhesion assays were conducted in serum-free M199
medium. About 5 × 104 cells were plated in
quadruplicate wells and, after 1 h of incubation, adherent cells
were washed, fixed in 1% glutaraldehyde for 10 min, stained with
crystal violet, lysed with 0.2% Triton X-100, and assayed by a
colorimetric test (36). The anti-adhesive assays were performed in a
similar way on fibronectin-coated plates. After blockage with 1% BSA,
the cells were added to the wells in the presence of increasing
concentration of endorepellin or endostatin. After 1 h of
incubation, the wells were treated as above.
1 G418 and
500 ng ml
1 puromycin), several endorepellin/AP-expressing
clones were identified using the Great EscAPeTM SEAP system
(Clontech), which detects AP. Briefly, conditioned
media from untransfected cells (negative control) and from stably
transfected cells secreting either AP (positive control) or
endorepellin/AP chimera were incubated at 65 °C for 30 min to
inactivate endogenous phosphatases, cooled on ice, and then mixed with
CSPD substrate/chemiluminescent enhancer for 10 min, followed by
exposure to x-ray film for 5-10 s. In addition, the nature of the AP
alone and the chimeric protein was identified following
immunoprecipitation with a mouse monoclonal antibody (Clone 8B6, Sigma)
against human placental AP linked to agarose beads. Binding studies
were performed using various cell lines incubated with 0.5 ml of
serum-free media conditioned by expressing or control 293-EBNA cells
for 48 h. After a 1.5-h incubation at 25 °C, the cells were
washed six times, lysed in 1% Triton X-100, 20 mM
Tris-HCl, pH 7.5, and processed as stated above.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase expression (Fig. 1c). To
corroborate the yeast interaction, we in vitro transcribed
and translated endorepellin and collagen XVIII (clone A3), showing the
~81- and ~65-kDa fragments, respectively (Fig. 1d). We
could co-precipitate the two proteins with an anti-HA antibody that
recognizes the oligopeptide epitope HA present at the C terminus of
collagen XVIII (Fig. 1d). To determine whether endostatin,
which is encoded by the C terminus of collagen type XVIII (Fig.
1b), could interact with endorepellin, we cloned the
endostatin sequence into pGADT7 vector, and then in vitro transcribed and translated the insert, which generated a 23-kDa band
(Fig. 1e, lane 3). As a further control, we
subcloned domain III of perlecan into the pGBKT7 vector and then
in vitro transcribed and translated the insert, which gave
the expected ~130-kDa peptide (Fig. 1e, lane
1). The results showed that only endorepellin interacted with
endostatin (Fig. 1e, lane 5). In contrast, domain
III of human perlecan did not bind (lane 4).
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Fig. 1.
Perlecan domain V (endorepellin) binds to the
anti-angiogenic factor endostatin. a, schematic
representation of human perlecan domain V/endorepellin which was used
as a bait in the yeast two-hybrid screening. This domain consists of
three laminin-type G (LG1-LG3) modules (orange ovals)
separated by four EGF-like (EG1-EG4) modules (blue
rectangles), in an arrangement highly conserved across species (2,
43). b, schematic representation of the human chain of
type XVIII collagen. The beginning of the clone A3 sequence (NCBI
accession no. AF018082), which interacted with endorepellin, is shown
in the top margin. The collagenous (triple-helical) and
non-collagenous domains are indicated by rods and blue
boxes, respectively. The C-terminal endostatin fragment is
highlighted in orange. c, growth and
-galactosidase activity triggered by the interaction of endorepellin
with collagen type XVIII fragment compared with the positive (p53 and
T-antigen) and negative controls (lamin and T-antigen). d,
co-immunoprecipitation of collagen XVIII (clone A3) and endorepellin
following in vitro transcription/translation using
[35S]methionine as the labeled precursor. Endorepellin
(lane 1) and the fragment of collagen XVIII (clone A3)
(lane 2) were mixed in equimolar amounts and
co-immunoprecipitated with either anti-HA (lane 3) or no
antibody (lane 4). e, co-immunoprecipitation of
endostatin with endorepellin. Domain III, a perlecan core protein
domain used as a negative control (lane 1), endorepellin
(lane 2), and endostatin (lane 3) were generated
by in vitro transcription/translation using
[35S]methionine as the labeled precursor. Endostatin,
which contains the HA tag at its C terminus, was mixed with either
domain III (lane 4) or endorepellin (lane 5) and
immunoprecipitated with anti-HA antibody. These experiments were
repeated three times with comparable results. f, solid-phase
binding assays of 125I-endorepellin as the soluble ligand
to endostatin-coated wells in the absence (
) or presence (
) of
25-molar excess of unlabeled endorepellin. The values represent the
mean ± S.E. of quadruplicate determinations. g,
solid-phase binding assays of soluble 125I-endorepellin to
either fibronectin (
) or collagen I (
) as the solid substrates.
The recombinant proteins were coated onto Immulon wells at 10 µg
ml
1 and then challenged with increasing concentrations of
125I-endorepellin (~1018 cpm
mol
1) as indicated. The values represent the
mean ± S.E. of quadruplicate determinations. These experiments
were repeated twice with comparable results.
1. We found a saturable binding of
125I-endorepellin to endostatin in the 60-70
nM range, with half maximal binding of ~48 nm (Fig.
1f). Specificity of binding was determined by competition
experiments with 25-molar excess of cold endorepellin (Fig.
1f). In contrast, endorepellin did not substantially bind to
either fibronectin or collagen type I (Fig. 1g).
1-
7) of domain
V/endorepellin (Fig. 2). Robust growth in
quadruple minus media was observed in cells co-transformed with
full-length endorepellin and
1 and
5, the only two mutants that
encompassed the LG2 module (Fig. 2a). These results were
corroborated by
- and
-galactosidase assays (Fig. 2b).
In addition to growth in amino acid-deficient media, transcription of
LacZ (
- and
-galactosidase) under the control of
distinct GAL4 upstream-activating sequences, and the subsequent ability of the co-transformant yeast strains to express functional galactosidase activity, provides an additional strong proof
of a true protein-protein interaction (47). Thus, the LG2 module of
endorepellin is likely to be the specific site of endostatin binding,
although in the native perlecan core protein, the role of the flanking
sequences remains to be established.
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Fig. 2.
Endostatin specifically interacts with the
LG2 module of endorepellin. a, schematic
representation of domain V and various deletion mutants. This domain
consists of three laminin-type G (LG1-LG3) modules (orange
ovals) separated by four EGF-like (EG1-EG4) modules (blue
rectangles) in an arrangement highly conserved across species (2,
43). The growth is indicated by semi-quantitative assessment with
maximal growth at +++. The numbers within parentheses designate the
amino acid position based on the mature protein core. b,
representative - and
-galactosidase assays of various deletion
mutants; pGB53/pGADT is the positive control.
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Fig. 3.
Recombinant endorepellin inhibits
VEGF-mediated chemotactic migration of endothelial cells.
a, purification of endorepellin from media conditioned by
293-EBNA cells expressing the 81-kDa endorepellin tagged with
His6. Coomassie-stained SDS-PAGE (left) and
Western immunoblotting with anti-His6 antibody
(right) of negative control media (lanes 1 and
4), flow through (lanes 2 and 5), and
250 mM imidazole eluate (lanes 3 and
6). b, Coomassie-stained SDS-PAGE of purified
endorepellin following elution from a DEAE Sephacel chromatography.
Lane 1, molecular weight rainbow markers; lane 2,
starting material; lane 3, 150 mM NaCl eluate;
lane 4, 2 M NaCl eluate. c and
d, HUVEC migration assays through fibrillar collagen type I
using 10 ng ml 1 VEGF as a chemotactic inducer with or
without incubation with various concentrations of endostatin
(ES) and endorepellin (ER) as indicated. SFM,
serum-free medium. The values represent the mean ± S.E. of
quadruplicate determinations. These experiments were repeated three
times with comparable results.
1 (12-120
nM) endorepellin (Fig. 3c). Interestingly,
endorepellin was more active than recombinant endostatin purified from
Pichia pastoris yeast cells. Subsequent dilution experiments
revealed that endorepellin was fully active at 0.5 µg
ml
1 (6 nM) (Fig. 3d), with a
calculated IC50 of 2 nM (± 0.1, n = 11). In some preparations, endorepellin was active
even at picomolar concentrations (not shown). These experiments were
repeated several times with various preparations of endorepellin, and a
marked suppression of HUVEC migration was consistently found. In
contrast to endostatin, the migratory response was not dependent on the preincubation of the endothelial cells with endorepellin. In
experiments where endorepellin was placed in the lower chambers of the
invasion assay, we found similar inhibition of VEGF-induced migration
(not shown).
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Fig. 4.
Recombinant endorepellin is a powerful
anti-angiogenic factor in vivo. a-c, CAM assays
3 days after the application of sponges (asterisk)
containing VEGF (10 ng), VEGF (10 ng) + endorepellin (400 ng), or
buffer alone. A total of 20 embryos were used for each experiment.
Scale bar, 1 mm. d and e, CAM assays
using sponges (asterisk) containing either 0.5 × 106 WiDr colon carcinoma cells alone or in combination with
3 µg of recombinant endorepellin. A total of 20 embryos were used for
each experiment. These experiments were repeated three times with
identical results. f and g, quantification of the
CAM assays. To quantify the volume of the newly formed vessels, ten
squares of ~500 µm2 each were randomly selected around
the sponge area. The mean vessel area was calculated using the NIH
Image software program (version 1.61) using at least four embryos per
experimental point. The values represent the mean ± S.E. of
quadruplicate determinations. These experiments were repeated three
times.
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Fig. 5.
Endorepellin blocks blood vessel ingrowth in
the Matrigel plug, and prevents endothelial tube formation induced by
fibrillar collagen. a and b, digital images
of dorsal skin viewed from the inside, 2 weeks after subcutaneous
injection of Matrigel supplemented with FGF2 and either BSA or
endorepellin. Notice the decreased neovascularization around the
Matrigel plug (asterisk) in the endorepellin-treated samples
as compared with the control samples (arrows). Scale
bars, 5 mm. c and d, photomicrographs of
Matrigel plugs from either control or endorepellin-treated samples,
respectively. The ingrowths of new blood vessels are markedly enhanced
in the control samples (arrows), as compared with the
endorepellin-treated samples. Scale bars = 200 µm.
e-h, gallery of light micrographs capturing the production
of HUVEC tube-like formation within a collagen type I matrix either
alone or following the addition of endorepellin, endostatin, or both.
Several concentrations of endorepellin and endostatin (50- 150 nM) were tested. In this assay, 4 × 105
cells were incubated for 24 h, and pictures were taken at various
intervals. The images shown are from the 4-h time point. The images at
24 h were identical to those obtained at 12 h (not shown),
indicating that the effects of endorepellin are long lasting. These
experiments were repeated three times with comparable results.
Scale bar, 250 µm.
1,
respectively) that gave suboptimal and optimal inhibition of HUVEC
migration. When endostatin and endorepellin were concurrently present,
there was an overall inhibition of their activities (Fig. 6, a and b). By
plotting the percentage of migrated cells derived from normalized data
of five independent experiments, against the increasing molar ratios of
endostatin/endorepellin, maximal neutralization was achieved at ~1:1
molar ratio. Thus, the combined effects of endostatin and endorepellin
are not additive, but they may lead to an attenuation of their
respective anti-angiogenic activities.
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Fig. 6.
Biological consequences of
endostatin/endorepellin interaction. a and
b, HUVEC migration assays through fibrillar collagen using
10 ng ml 1 VEGF as a chemotactic inducer and preincubation
of HUVECs for 30 min with various concentrations of endostatin
(ES), endorepellin (ER), or various combinations
as indicated. The values are presented as the percentage of maximal
migration induced by VEGF, arbitrarily set at 100%. Panel a
shows the summary of three independent experiments run in
quadruplicate, mean ± S.E. The values in panel b
derive from an additional experiment run in quadruplicate, mean ± S.E. SFM, serum-free medium.
1) and found the predominant
81-kDa band, with a small fraction of labeling going into a 25-kDa
fragment (Fig. 7a). To
establish the nature of this fragment, we transferred a similar
preparation to polyvinylidene difluoride membrane and sequenced the N
terminus. This confirmed that the 25-kDa fragment encompassed nearly
all the LG3 module, with a specific cleavage between Asn-4196 and Asp-4197 (Fig. 7a). Covalent affinity cross-linking
experiments using a membrane-impermeable cross-linker (BS3)
revealed a major complex of very high Mr, which
did not penetrate the 7.5% SDS-PAGE (Fig. 7b).
Interestingly, the exogenously added LG3 module (the 25-kDa band in
Fig. 7b) was not cross-linked to HUVEC surface proteins,
suggesting that this part of endorepellin is dispensable for
binding.
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Fig. 7.
High affinity binding sites for endorepellin
on endothelial cells. a, autoradiography on a 10%
SDS-PAGE of endorepellin (ER) labeled to high specific
activity (~ 1018 cpm mol 1) using Iodogen
(Pierce). The autoradiograph was purposefully overexposed to show the
minor contaminant band of ~25 kDa. About 15 pmol of the purified
25-kDa band were electroblotted onto a Problott-polyvinylidene
difluoride membrane and microsequenced. The first N-terminal amino acid
residue is Asp-4197, near the beginning of the LG3 module. The seven
amino acid residues match perfectly to the human sequence of perlecan
(7, 8). b, covalent affinity cross-linking. HUVECs were
incubated with various concentrations of 125I-endorepellin
for 2 h as indicated in the bottom, and then incubated
for 30 min with 2 mM BS3, a
membrane-impermeable cross-linker. The reaction was terminated with 1 M Tris, pH 7.5, the cross-linked material was separated on
7.5% SDS-PAGE, and visualized by autoradiography. Notice that
endorepellin, but not the LG3 module, is complexed with high
Mr material that does not penetrate the
separating gel. c, saturation binding of
125I-endorepellin on HUVECs. Confluent HUVECs in 24-well
plates were incubated with increasing concentrations of
125I-endorepellin for 2.5 h at 4 °C in M199
containing 0.1% BSA, washed several times, and extracted in the
presence of protease inhibitors (41). Values represent the mean ± S.E. of three independent experiments run in triplicate. Nonspecific
binding was subtracted from the observed values. d,
Scatchard analysis of the data presented in c. Estimates of
receptor affinity and total binding capacity were made with the Wizard
program in the Sigma Plot 5.0 software package. These experiments were
repeated three times with similar results. e, displacement
of HUVEC bound 125I-endorepellin by increasing amounts of
cold endorepellin. The data represent the mean ± S.E. of two
individual experiments run in triplicate. In these experiments,
confluent HUVECs (~105 cells/dish) were incubated with
125I-endorepellin (5 nM) plus increasing
concentrations of recombinant unlabeled endorepellin, as indicated. The
cells were incubated at 4 °C for 3 h, washed three times,
extracted, and counted in total.
1 with a
Kd of 11 nM. Specificity of the binding
was further demonstrated by the efficient displacement of the
HUVEC-bound 125I-endorepellin by increasing amounts of cold
endorepellin (Fig. 7e), with IC50 of 27 nM, in good agreement with the binding isotherm shown
above. These experiments were repeated three times with comparable
results. Thus, we conclude that HUVECs possess a significant number of
high affinity endorepellin binding sites.
1 48 h
1. Immunoprecipitation studies using an anti-AP
monoclonal antibody linked to agarose showed the predicted sizes of
~141 kDa for the AP and endorepellin/AP chimera, respectively (Fig.
8b). Notably, incubation of endorepellin/AP-conditioned
media with HUVEC, MCF7, HT1080, and WiDr cells showed significant
binding to the cell surface of all the cells, with the highest binding
in WiDr followed by HUVEC, HT1080, and MCF7 (Fig. 8c). No
binding was observed with AP alone (not shown), further indicating the
specificity of the interaction between soluble endorepellin and the
cell surface. We also tested A431 squamous carcinoma cells, but we
could not block the endogenous AP even after 1-2 h incubation at
65 °C, indicating that these cells possess an endogenous heat-stable AP. To bypass this point, we performed binding studies of A431 and MCF7
(as a further control) cells using 125I-endorepellin as the
soluble ligand. The results showed a saturable binding for both A431
and MCF7 cells (Fig. 8, d and f). Scatchard analysis revealed a single receptor population for both A431 and MCF7
cells consisting of ~9 and ~7.5 × 104 sites
cell
1, with Kd values of 6.6 and
26 nM, respectively (Fig. 8, e and g). The lower number of receptors on the MCF7 as compared
with the HUVECs is in full agreement with the binding studies shown in
panel c. Notably, cross-linking experiments of A431 and MCF7 cells also showed the presence of high Mr
complexes that did not penetrate the gel (not shown) suggesting that
similar putative receptors are also present in these two cells.
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Fig. 8.
High affinity binding sites for endorepellin
on various tumor cell lines. a, generation of a
cellular system secreting either AP or endorepellin/AP chimeric
protein. Lanes 1 and 2 represent conditioned
media from either untransfected or AP-transfected 293-EBNA cells.
Lanes 3-13 represent media from positive and negative
clones of 293-EBNA cells stably transfected with the endorepellin/AP
construct. Conditioned media were incubated at 65 °C for 30 min to
inactivate endogenous phosphatases, cooled on ice, and then mixed with
CSPD substrate/chemiluminescent enhancer for 10 min (Great EscAPeTM
SEAP system, Clontech), followed by exposure to
x-ray film for 5-10 s. One of the strongest clone, (clone 6, lane 4) was amplified and used in the subsequent analyses in
b and c. b, identification of AP alone
or the chimeric endorepellin/AP protein following immunoprecipitation
with a mouse monoclonal antibody (Clone 8B6, Sigma) against human
placental AP linked to agarose beads. Coomassie Blue-stained 10%
SDS-PAGE of 0.5 ml of conditioned media from control (lane
2), AP-secreting (lane 3), or endorepellin/AP-secreting
(lane 4) 293-EBNA cells following incubation with 4 µl of
antibody-agarose resin. Because 1 ml of settled resin binds at least
0.5 mg of human placental AP protein, we estimate that 105
clone 6 cells express ~4 µg ml 1 48 h
1.
Molecular weight markers are in lane 1. c,
binding of endorepellin/AP chimeric protein to various cells. Binding
studies were performed using various cell lines (as indicated)
incubated with 0.5 ml of serum-free media conditioned by expressing or
control 293-EBNA cells for 48 h, using various dilutions as
indicated in the left margin. After a 1.5-h incubation at
25 °C, the cells were washed six times, lysed in 1% Triton X-100,
20 mM Tris- HCl, pH 7.5, and processed for AP assays as
above. d and f, saturation binding curves of
125I-endorepellin on A431 squamous carcinoma (d)
and MCF7 breast carcinoma (f) cells. Confluent cells in
24-well plates were incubated with increasing concentrations of
125I-endorepellin for 2.5 h at 4 °C in M199
containing 0.1% BSA, washed several times and extracted in the
presence of protease inhibitors (41). Values represent the mean ± S.E. of two independent experiments both run in quadruplicate.
Nonspecific binding was subtracted from the observed values.
e and g, Scatchard analyses of the data presented
in d and f, respectively. Estimates of receptor
affinity and total binding capacity were made with the Wizard program
in the Sigma Plot 5.0 software package.
Binding of various cells to fibronectin, collagen type I, and
endorepellin
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Fig. 9.
Endorepellin is counter-adhesive for
endothelial, fibrosarcoma and colon carcinoma cells. a,
gallery of light micrographs of crystal violet-stained HUVECs adhered
to fibronectin following incubation with endorepellin at the indicated
concentrations. Briefly, the cells were trypsinized and plated onto
fibronectin precoated (50 nM) wells in the presence of
increasing concentrations of endorepellin or in the presence of
phosphate-buffered saline (control). The cells were then
incubated for 1 h, washed, and stained with crystal violet. After
washing again, the cells were solubilized with Triton X-100, and the OD
at 600 nm was determined. The adhesion assays were conducted in
serum-free M199 medium. Scale bar, 100 µm. b
and c, displacement of HT1080 ( ) and WiDr (
) cells
from fibronectin-coated wells with increasing concentrations of either
endorepellin or endostatin, respectively. The calculated
IC50 values for HT1080 and WiDr were 110 and 40 nM, respectively. The values represent the mean ± S.E. (n = 4).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1) of endorepellin binding sites on HUVECs, with a
relatively high affinity constant (Kd = 11 nM). The specificity of binding was proved by the efficient
displacement of the HUVEC-bound 125I-endorepellin by
increasing amounts of cold endorepellin, with an IC50 of 27 nM, in good agreement with the affinity constant mentioned
above. The presence of putative endorepellin receptor(s) was further
corroborated by the presence of high Mr
complexes cross-linked to endorepellin. We also found high affinity
binding sites on A431 and MCF7 tumor cells using radioligand binding
assays similar to those used for HUVECs.
1 of urine (70).
Fourth, we have recently discovered an additional proteolytic cleavage
site (between Gly-3774 and Asp-3775) within the LG1 subdomain that
leads to the release of almost the entire endorepellin lacking only the
first 88 amino acid
residues.3 While this has not
been proven to occur in tissues, it is plausible to take place because
of the specificity of the cleavage site and the relatively high amounts
of this fragment that we obtained after purification in which the
mixture of protease inhibitors was suboptimal. Circulating forms of
endorepellin may be involved in the homeostatic control of angiogenesis
as previously proposed for endostatin, whose levels can reach 0.3 mg
liter
1 of blood (56). We would like to put forward a
provocative hypothesis, that is, it has been nearly three decades since
it was shown that extracts of cartilage contain potent anti-angiogenic
factors (71, 72), and because perlecan is highly expressed in
cartilage, both during development and adulthood (9, 29, 73),
endorepellin could conceivably be generated from the active remodeling
of cartilage that occurs during normal aging, inflammation or any other
condition that leads to cartilage turnover.
1 integrin and
-dystroglycan,
both of which have been shown to interact with perlecan domain V (36, 75, 76). In the case of
-dystroglycan, perlecan domain V was the
strongest ligand (Kd of 3 nM) and
required LG1 and LG2 modules, whereas LG3 module by itself had much
lower affinity (76). In agreement with these in vitro
binding assays, perlecan and
-dystroglycan co-localize at the
neuromuscular junctions (77, 78) where they may serve as cell-surface
acceptors for acetylcholinesterase. Interaction between perlecan and
-dystroglycan, together with laminin, may also play a key role in
the assembly of basement membranes during early development (74).
Experiments are underway to address these important issues.
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ACKNOWLEDGEMENTS |
---|
We thank R. Oldershaw and D. Birk for help with the CAM assays, J. Whitelock for valuable reagents, C. C. Clark for critical reading of the article, K. J. Williams for valuable advice, and S. Campbell for expert technical assistance.
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FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grants CA47282 and CA39481 (to R. V. I.), by a fellowship from the American-Italian Cancer Foundation, New York (to M. M.), and by National Institutes of Health Grant HL53590 and American Heart Association Grant 9910067U (to J. D. S.).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.
To whom correspondence should be addressed: Dept. of
Pathology, Anatomy and Cell Biology, Rm. 249 Jefferson Alumni Hall,
Thomas Jefferson University, 1020 Locust St., Philadelphia, PA 19107. E-mail: iozzo@lac.jci.tju.edu.
Published, JBC Papers in Press, November 14, 2002, DOI 10.1074/jbc.M210445200
2 M. Mongiat and R. V. Iozzo, unpublished observations.
3 J. Fu and R. V. Iozzo, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are: FGF2, fibroblast growth factor 2; LG, laminin-G like module; CAM, chorioallantoic membrane; HUVEC, human umbilical vein endothelial cells; VEGF, vascular endothelial growth factor; BSA, bovine serum albumin; AP, alkaline phosphatase; HA, hemagglutinin; NTA, nitrilotriacetic acid; ELISA, enzyme-linked immunosorbent assay; BS3, bis-sulfosuccimidyl-suberate.
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REFERENCES |
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1. | Hassell, J. R., Robey, P. G., Barrach, H. J., Wilczek, J., Rennard, S. I., and Martin, G. R. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 4494-4498[Abstract] |
2. | Dunlevy, J. R., and Hassell, J. R. (2000) in Proteoglycans; Structure, biology and molecular interactions (Iozzo, R. V., ed) , pp. 275-326, Marcel Dekker, Inc., New York |
3. | Iozzo, R. V. (1998) Annu. Rev. Biochem. 67, 609-652[CrossRef][Medline] [Order article via Infotrieve] |
4. |
Yurchenco, P. D.,
Cheng, Y.-S.,
and Ruben, G. C.
(1987)
J. Biol. Chem.
262,
17668-17676 |
5. | Yurchenco, P. D., and O'Rear, J. J. (1994) Curr. Opin. Cell Biol. 6, 674-681[Medline] [Order article via Infotrieve] |
6. |
Noonan, D. M.,
Fulle, A.,
Valente, P.,
Cai, S.,
Horigan, E.,
Sasaki, M.,
Yamada, Y.,
and Hassell, J. R.
(1991)
J. Biol. Chem.
266,
22939-22947 |
7. | Kallunki, P., and Tryggvason, K. (1992) J. Cell Biol. 116, 559-571[Abstract] |
8. |
Murdoch, A. D.,
Dodge, G. R.,
Cohen, I.,
Tuan, R. S.,
and Iozzo, R. V.
(1992)
J. Biol. Chem.
267,
8544-8557 |
9. | Handler, M., Yurchenco, P. D., and Iozzo, R. V. (1997) Dev. Dyn. 210, 130-145[CrossRef][Medline] [Order article via Infotrieve] |
10. |
Iozzo, R. V.,
and San Antonio, J. D.
(2001)
J. Clin. Invest.
108,
349-355 |
11. | Arikawa-Hirasawa, E., Watanabe, E., Takami, H., Hassell, J. R., and Yamada, Y. (1999) Nat. Genet. 23, 354-358[CrossRef][Medline] [Order article via Infotrieve] |
12. |
Costell, M.,
Gustafsson, E.,
Aszódi, A.,
Mörgelin, M.,
Bloch, W.,
Hunziker, E.,
Addicks, K.,
Timpl, R.,
and Fässler, R.
(1999)
J. Cell Biol.
147,
1109-1122 |
13. |
Costell, M.,
Carmona, R.,
Gustafsson, E.,
González-Iriarte, M.,
Fässler, R.,
and Munoz-Chápuli, R.
(2002)
Circ. Res.
91,
158-164 |
14. | Nugent, M. A., Karnovsky, M. J., and Edelman, E. R. (1993) Circ. Res. 73, 1051-1060[Abstract] |
15. | Weiser, M. C. M., Grieshaber, N. A., Schwartz, P. E., and Majack, R. A. (1997) Mol. Biol. Cell 8, 999-1011[Abstract] |
16. | Aviezer, D., Hecht, D., Safran, M., Eisinger, M., David, G., and Yayon, A. (1994) Cell 79, 1005-1013[Medline] [Order article via Infotrieve] |
17. |
Whitelock, J. M.,
Murdoch, A. D.,
Iozzo, R. V.,
and Underwood, P. A.
(1996)
J. Biol. Chem.
271,
10079-10086 |
18. |
Nugent, M. A.,
Nugent, H. M.,
Iozzo, R. V.,
Sanchack, K.,
and Edelman, E. R.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
6722-6727 |
19. | Iozzo, R. V. (1984) J. Cell Biol. 99, 403-417[Abstract] |
20. | Tapanadechopone, P., Tumova, S., Jiang, X., and Couchman, J. R. (2001) Biochem. J. 355, 517-527[CrossRef][Medline] [Order article via Infotrieve] |
21. | Cohen, I. R., Murdoch, A. D., Naso, M. F., Marchetti, D., Berd, D., and Iozzo, R. V. (1994) Cancer Res. 54, 5771-5774[Abstract] |
22. | Iozzo, R. V., Cohen, I. R., Grässel, S., and Murdoch, A. D. (1994) Biochem. J. 302, 625-639[Medline] [Order article via Infotrieve] |
23. | Aviezer, D., Iozzo, R. V., Noonan, D. M., and Yayon, A. (1997) Mol. Cell. Biol. 17, 1938-1946[Abstract] |
24. | Adatia, R., Albini, A., Carlone, S., Giunciuglio, D., Benelli, R., Santi, L., and Noonan, D. M. (1998) Ann. Oncol. 8, 1257-1261[Abstract] |
25. |
Sharma, B.,
Handler, M.,
Eichstetter, I.,
Whitelock, J.,
Nugent, M. A.,
and Iozzo, R. V.
(1998)
J. Clin. Invest.
102,
1599-1608 |
26. |
Mongiat, M.,
Taylor, K.,
Otto, J.,
Aho, S.,
Uitto, J.,
Whitelock, J.,
and Iozzo, R. V.
(2000)
J. Biol. Chem.
275,
7095-7100 |
27. | Ghiselli, G., Eichstetter, I., and Iozzo, R. V. (2001) Biochem. J. 359, 153-163[CrossRef][Medline] [Order article via Infotrieve] |
28. | Timpl, R. (1993) Experientia 49, 417-428[Medline] [Order article via Infotrieve] |
29. |
SundarRaj, N.,
Fite, D.,
Ledbetter, S.,
Chakravarti, L.,
and Hassell, J. R.
(1995)
J. Cell Sci.
108,
2663-2672 |
30. | Klein, G., Conzelmann, S., Beck, S., Timpl, R., and Müller, C. A. (1995) Matrix Biol. 14, 457-465[CrossRef][Medline] [Order article via Infotrieve] |
31. | Gauer, S., Schulzelohoff, E., Schleicher, E., and Sterzel, R. B. (1996) Eur. J. Cell Biol. 70, 233-242[Medline] [Order article via Infotrieve] |
32. | Whitelock, J. M., Graham, L. D., Melrose, J., Murdoch, A. D., Iozzo, R. V., and Underwood, P. A. (1999) Matrix Biol. 18, 163-178[CrossRef][Medline] [Order article via Infotrieve] |
33. | Folkman, J., and D'Amore, P. A. (1996) Cell 87, 1153-1155[Medline] [Order article via Infotrieve] |
34. |
Iozzo, R. V.
(2001)
J. Clin. Invest.
108,
165-167 |
35. |
Esko, J. D.,
and Lindahl, U.
(2001)
J. Clin. Invest.
108,
169-173 |
36. | Brown, J. C., Sasaki, T., Göhring, W., Yamada, E., and Timpl, R. (1997) Eur. J. Biochem. 250, 39-46[Abstract] |
37. | Friedrich, M. V. K., Göhring, W., Mörgelin, M., Brancaccio, A., David, G., and Timpl, R. (1999) J. Mol. Biol. 294, 259-270[CrossRef][Medline] [Order article via Infotrieve] |
38. |
Mongiat, M.,
Otto, J.,
Oldershaw, R.,
Ferrer, F.,
Sato, J. D.,
and Iozzo, R. V.
(2001)
J. Biol. Chem.
276,
10263-10271 |
39. | Montesano, R., Orci, L., and Vassalli, P. (1983) J. Cell Biol. 97, 1648-1652[Abstract] |
40. | Auerbach, R., Akhtar, N., Lewis, R. L., and Shinners, B. L. (2000) Cancer Metastasis Rev. 19, 167-172[CrossRef][Medline] [Order article via Infotrieve] |
41. |
Santra, M.,
Eichstetter, I.,
and Iozzo, R. V.
(2000)
J. Biol. Chem.
275,
35153-35161 |
42. |
Santra, M.,
Reed, C. C.,
and Iozzo, R. V.
(2002)
J. Biol. Chem.
277,
35671-35681 |
43. | Timpl, R., Tisi, D., Talts, J. F., Andac, Z., Sasaki, T., and Hohenester, E. (2000) Matrix Biol. 19, 309-317[CrossRef][Medline] [Order article via Infotrieve] |
44. | O'Reilly, M. S., Boehm, T., Shing, Y., Fukai, N., Vasios, G., Lane, W. S., Flynn, E., Birkhead, J. R., Olsen, B. R., and Folkman, J. (1997) Cell 88, 277-285[Medline] [Order article via Infotrieve] |
45. |
Halfter, W.,
Dong, S.,
Schurer, B.,
and Cole, G. J.
(1998)
J. Biol. Chem.
273,
25404-25412 |
46. |
Saarela, J.,
Rehn, M.,
Oikarinen, A.,
Autio-Harmainen, H.,
and Pihlajaniemi, T.
(1998)
Am. J. Pathol.
153,
611-626 |
47. | Fields, S., and Sternglanz, R. (1994) Trends Genet. 10, 286-292[CrossRef][Medline] [Order article via Infotrieve] |
48. | Tapanadechopone, P., Hassell, J. R., Rigatti, B., and Couchman, J. R. (1999) Biochem. Biophys. Res. Commun. 265, 680-690[CrossRef][Medline] [Order article via Infotrieve] |
49. |
Friedrich, M. V. K.,
Schneider, M.,
Timpl, R.,
and Baumgartner, S.
(2000)
Eur. J. Biochem.
267,
3149-3159 |
50. |
Yamaguchi, N.,
Anand-Apte, B.,
Lee, M.,
Sasaki, T.,
Fukai, N.,
Shapiro, R.,
Que, I.,
Lowik, C.,
Timpl, R.,
and Olsen, B. R.
(1999)
EMBO J.
18,
4414-4423 |
51. | Risau, W. (1997) Nature 386, 671-674[CrossRef][Medline] [Order article via Infotrieve] |
52. | Sage, E. H. (1997) Trends Cell Biol. 7, 182-186[CrossRef] |
53. | Santra, M., Skorski, T., Calabretta, B., Lattime, E. C., and Iozzo, R. V. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7016-7020[Abstract] |
54. | Folkman, J., Haudenschild, C. C., and Zetter, B. R. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 5217-5221[Abstract] |
55. | Flanagan, J. G., and Leder, P. (1990) Cell 63, 185-194[Medline] [Order article via Infotrieve] |
56. |
Sasaki, T.,
Fukai, N.,
Mann, K.,
Göhring, W.,
Olsen, B. R.,
and Timpl, R.
(1998)
EMBO J.
17,
4249-4256 |
57. | Sasaki, T., Larsson, H., Tisi, D., Claesson-Welsh, L., Hohenester, E., and Timpl, R. (2000) J. Mol. Biol. 301, 1179-1190[CrossRef][Medline] [Order article via Infotrieve] |
58. |
Chang, Z.,
Choon, A.,
and Friedl, A.
(2000)
Am. J. Pathol.
155,
71-76 |
59. |
Knox, S.,
Merry, C.,
Stringer, S.,
Melrose, J.,
and Whitelock, J.
(2002)
J. Biol. Chem.
277,
14657-14665 |
60. | Villar, M. J., Hassell, J. R., and Brandan, E. (1999) J. Cell. Biochem. 75, 665-674[CrossRef][Medline] [Order article via Infotrieve] |
61. | Lundmark, K., Tran, P. K., Kinsella, M. G., Clowes, A. W., Wight, T. N., and Hedin, U. (2001) J. Cell. Physiol. 188, 67-74[CrossRef][Medline] [Order article via Infotrieve] |
62. | Mathiak, M., Yenisey, C., Grant, D. S., Sharma, B., and Iozzo, R. V. (1997) Cancer Res. 57, 2130-2136[Abstract] |
63. |
Kuo, C. J.,
LaMontagne, K. R.,
Garcia-Cardena, G.,
Ackley, B. D.,
Kalman, D.,
Park, S.,
Christofferson, R.,
Kamihara, J.,
Ding, Y.-H., Lo, K.-M.,
Gillies, S.,
Folkman, J.,
Mulligan, R. C.,
and Javaherian, K.
(2001)
J. Cell Biol.
152,
1233-1246 |
64. |
Ackley, B. D.,
Crew, J. R.,
Elamaa, H.,
Pihlajaniemi, T.,
Kuo, C. J.,
and Kramer, J. M.
(2001)
J. Cell Biol.
152,
1219-1232 |
65. | Hanahan, D., and Folkman, J. (1996) Cell 86, 353-364[Medline] [Order article via Infotrieve] |
66. | Marneros, A. G., and Olsen, B. R. (2001) Matrix Biol. 20, 337-345[CrossRef][Medline] [Order article via Infotrieve] |
67. |
Xu, J.,
Rodriguez, D.,
Petitclerc, E.,
Kim, J. J.,
Hangai, M.,
Yuen, S. M.,
Davis, G. E.,
and Brooks, P. C.
(2001)
J. Cell Biol.
154,
1069-1079 |
68. |
Roghani, M.,
and Moscatelli, D.
(1992)
J. Biol. Chem.
267,
22156-22162 |
69. |
Smirnov, S. P.,
McDearmon, E. L., Li, S.,
Ervasti, J. M.,
Tryggvason, K.,
and Yurchenco, P. D.
(2002)
J. Biol. Chem.
277,
18928-18937 |
70. | Oda, O., Shinzato, T., Ohbayashi, K., Takai, I., Kunimatsu, M., Maeda, K., and Yamanaka, N. (1996) Clin. Chim. Acta 255, 119-132[CrossRef][Medline] [Order article via Infotrieve] |
71. |
Folkman, J.,
and Shing, Y.
(1992)
J. Biol. Chem.
267,
10931-10934 |
72. | Folkman, J. (1995) Nat. Med. 1, 27-31[Medline] [Order article via Infotrieve] |
73. |
French, M. M.,
Smith, S. E.,
Akanbi, K.,
Sanford, T.,
Hecht, J.,
Farach-Carson, M. C.,
and Carson, D. D.
(1999)
J. Cell Biol.
145,
1103-1115 |
74. |
Henry, M. D.,
Satz, J. S.,
Brakebusch, C.,
Costell, M.,
Gustafsson, E.,
Fässler, R.,
and Campbell, K. P.
(2001)
J. Cell Sci.
114,
1137-1144 |
75. | Hayashi, K., Madri, J. A., and Yurchenco, P. D. (1992) J. Cell Biol. 119, 945-959[Abstract] |
76. |
Talts, J. F.,
Andac, Z.,
Göhring, W.,
Brancaccio, A.,
and Timpl, R.
(1999)
EMBO J.
18,
863-870 |
77. | Peng, H. B., Ali, A. A., Daggett, D. F., Rauvala, H., Hassell, J. R., and Smalheiser, N. R. (1998) Cell Adhesion Comm. 5, 475-489[Medline] [Order article via Infotrieve] |
78. |
Peng, H. B.,
Xie, H.,
Rossi, S. G.,
and Rotundo, R. L.
(1999)
J. Cell Biol.
145,
911-921 |