From the Departments of Molecular Genetics and
¶ Pharmacology, College of Medicine, University of
Illinois, Chicago, Illinois 60607-7170
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ABSTRACT |
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Cyr61 is a member of a family of growth
factor-inducible immediate-early gene products thought to act
cooperatively with the activities of growth factors. Upon synthesis,
Cyr61 is secreted and is predominantly incorporated into the
extracellular matrix. Recently, we demonstrated that Cyr61 promotes
cell adhesion and migration and augments growth factor-induced DNA
synthesis (Kireeva, M. L., Mo, F.-E., Yang, G. P., and Lau,
L. F. (1996) Mol. Cell. Biol. 16, 1326-1334). In the
present study, we investigated possible candidate receptor(s) on human
umbilical vein endothelial cells (HUVECs) mediating adhesion to Cyr61.
Under both serum-containing and serum-free conditions, adhesion of
HUVECs to Cyr61 was dose-dependent, saturable, and
abolished by affinity-purified anti-Cyr61 antibodies. Cell adhesion to
Cyr61 was divalent cation-dependent and specifically inhibited by the peptide RGDS and LM609, a monoclonal antibody against
integrin v
3. Furthermore, purified
v
3 bound directly to an affinity matrix
of Cyr61-coupled Sepharose 4B, and this interaction was specifically
blocked by anti-Cyr61 antibodies. Additionally, in a solid phase
binding assay, soluble Cyr61 bound to immobilized
v
3 in a dose-dependent
manner, and half-saturation binding occurred at approximately 5 nM Cyr61. As expected, the interaction of Cyr61 with
immobilized
v
3 was blocked by RGDS and
LM609. In sum, these results identified Cyr61 as a novel ligand for
v
3 and indicate that the adhesion of
HUVECs to Cyr61 is mediated through interaction with this integrin. The
possibility that integrin
v
3 functions as
a signaling receptor for Cyr61 accounts for most if not all activities
that can be ascribed to Cyr61 to date and suggests a mechanism of
action discussed herein.
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INTRODUCTION |
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One of the primary cellular responses to the actions of polypeptide growth factors is the rapid and transient activation of a set of immediate-early genes, the expression of which is thought to be involved in mediating the biological responses of the growth factors (1, 2). Among the immediate-early genes identified in fibroblasts is cyr61 (3), which encodes a secreted cysteine-rich and heparin-binding protein associated with the extracellular matrix (ECM)1 or with the cell surface (4). In a recent study, we reported the purification of recombinant Cyr61 and its biological activities in cell culture systems. Thus, Cyr61 promotes cell adhesion, stimulates cell migration, and augments growth factor-induced DNA synthesis (5). Fisp12, a closely related protein also encoded by an immediate-early gene in fibroblasts (6), has indistinguishable activities (7). During embryogenesis, Cyr61 exhibits a temporally restricted and tissue-specific distribution that closely associates with the development of cartilage and the circulatory system (8). Consistent with this expression pattern, Cyr61 promotes the differentiation of mouse limb bud mesenchymal cells into chondrocytes in micromass cultures (9). Taken together, these findings suggest that Cyr61 functions as a downstream regulator of growth factor actions, possibly by acting as an ECM-associated signaling molecule to regulate physiological processes involving cell adhesion, migration, proliferation, and differentiation.
The biological activities of Cyr61 suggest that it might interact with
a cell surface receptor. The ability of Cyr61 to mediate cell adhesion
and a strict requirement for divalent cations in this process (5) are
consistent with the possible interaction of Cyr61 with one of the
divalent cation-dependent cell adhesion receptors of the
integrin, selectin, or cadherin families (10-13). Selectins and
cadherins are primarily involved in cell-cell interactions. Integrins,
on the other hand, are a large family of heterodimeric adhesion
receptors involved in both cell-cell and cell-ECM interactions (11). In
addition to serving as cell adhesion receptors, integrins also play an
essential role in cell motility (14, 15). Furthermore, integrin
occupancy and clustering induces signaling cascades that intersect with
growth factor-induced signaling processes that regulate cell
proliferation and differentiation (16-21). Based on these
considerations, we hypothesized that Cyr61 might interact with an
integrin receptor, thereby promoting cell adhesion and migration as
well as augmenting growth factor-induced DNA synthesis. In the present
study, by examining the effect of inhibitors of integrin
v
3 on cell adhesion and in
vitro protein binding assays, we investigated the possibility that
adhesion of HUVECs to Cyr61 is mediated through direct interaction of
Cyr61 and integrin
v
3.
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MATERIALS AND METHODS |
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Antibodies and Peptides--
The
anti-v
3 monoclonal antibody LM609 (22)
was a generous gift of Dr. D. A. Cheresh (Scripps Research
Institute, La Jolla, CA). The monoclonal antibodies LM142
(anti-
v) and JBS5
(anti-
5
1) were purchased from Chemicon
(Temeculo, CA). Production and characterization of polyclonal
anti-
3 antibodies have been described previously (23).
Polyclonal anti-Cyr61 antibodies were raised in rabbits and affinity
purified as described previously (3). Anti-sera against human and
bovine vitronectin were from Life Technologies, Inc. (Gaithersburg,
MD), and human plasma vitronectin was from Collaborative Biomedical
Products. Horseradish peroxidase-conjugated goat anti-rabbit antibody
was obtained from KPL (Gaithersburg, MD).
Cell Culture-- HUVECs (ATCC, starting passage 12) were grown in complete F12K medium (Sigma) containing 10% fetal bovine serum, 2 mM glutamine, 100 µg/ml heparin, and 30 µg/ml endothelial cell growth supplement (Collaborative Biomedical Products, Bedford, MA). Cells were passaged 1:4 twice a week and used in experiments at passages 18-23. Sf9 insect cells were passaged and maintained in serum-free Sf900-II medium (Life Technologies, Inc.).
Protein Purifications-- Recombinant Cyr61, synthesized in a baculovirus expression system using Sf9 insect cells, was purified from serum-free conditioned media by chromatography on Sepharose S as described (5, 7). SDS-polyacrylamide gel electrophoresis analysis of purified Cyr61 revealed the presence of a single Coomassie Blue-stained 40-kDa protein with the predicted molecular mass of Cyr61. As expected, the purified protein was immunoblotted by the anti-Cyr61 antibodies (Fig. 1A).
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Cell Adhesion Assay-- Adhesion of HUVECs in serum-containing complete medium to Cyr61 immobilized onto microtiter wells was performed as described (5, 7). In some experiments, HUVECs were harvested with 2 mM EDTA in PBS (20 min, room temperature), washed twice with F12K serum-free medium containing 7 mg/ml BSA (Sigma) and resuspended in the same medium at 2.5 × 105 cells/ml. Where indicated, EDTA, inhibitory peptides, and antibodies were preincubated with the cells for 15 min before plating. After incubation at 37 °C for 20 min, the wells were washed with PBS, and adherent cells were fixed and stained with methylene blue. HUVEC adhesion was quantitated by dye extraction and measurement of absorbance at 620 nm.
Binding of Integrin v
3 to
Cyr61-coupled Sepharose--
Purified Cyr61 protein (0.6 mg/ml) or
human plasma vitronectin (1 mg/ml; Collaborative Biomedical Research)
was coupled to cyanogen bromide-activated Sepharose 4B (hydrated bead
volume, 100 µl; Pharmacia) using standard procedures as recommended
by the manufacturer. After blocking nonspecific sites with 1% BSA, the
affinity matrices were equilibrated with TBS-OG (50 mM
Tris, pH 7.5, 150 mM NaCl containing 1 mM
CaCl2, 1 mM MgCl2, 0.1 mM phenylmethanesulfonyl fluoride, and 5 mM
octylglucoside). Purified integrin
v
3
(250 ng in 50 µl TBS-OG) was incubated with 20 µl of Cyr61- or
vitronectin-coupled Sepharose beads overnight at 4 °C. Where
indicated, anti-Cyr61 or anti-human vitronectin antisera were incubated
with the ligand-coupled Sepharose for 2 h at 4 °C prior to the
addition of integrin
v
3. Following
extensive washing with TBS-OG, bound integrin was extracted with
nonreducing SDS gel loading buffer, resolved by SDS-polyacrylamide gel
electrophoresis, and detected by immunoblotting with
anti-
3 polyclonal antibodies.
Solid Phase Binding Assay of Cyr61 to Integrin
v
3--
The binding of Cyr61 to
immobilized
v
3 was measured using
previously described methods with slight modifications (26, 27).
Briefly, microtiter wells (Pro-Bind plates, Falcon) were coated with
purified
v
3 (1 µg/ml, 50 µl/well)
overnight at 4 °C, and then blocked with 2% BSA for 2 h at
room temperature. After four washes with PBS, pH 7.5, containing 1 mM CaCl2, 1 mM MgCl2, and 5 mM octylglucoside, Cyr61 or vitronectin was added and
incubated for 3 h at room temperature. In inhibition studies,
EDTA, blocking peptides and antibodies were preincubated with the
immobilized integrin for 1 h before the addition of ligands to
wells containing the inhibitors. Bound ligands were detected by
specific polyclonal antisera diluted in PBS, pH 7.5, containing 1 mM CaCl2,1 mM MgCl2, and 5 mg/ml BSA (anti-Cyr61, 1:500; anti-vitronectin, 1:1000) followed
by horseradish peroxidase-conjugated secondary antibody (1:20,000). The
color reaction was developed using the horseradish peroxidase
immunoassay kit (Bio-Rad), and the absorbance at 450 nm was
measured.
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RESULTS |
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Adhesion of HUVECs to Cyr61 Is Dependent on Integrin
v
3--
In a previous report (5), we
showed that HUVECs, suspended in a serum-containing medium, adhere to
immobilized Cyr61 in a divalent cation-dependent manner,
suggesting that the observed adhesion is mediated through a
divalent-cation dependent adhesion receptor. However, inasmuch as serum
contains several adhesive proteins, it remains a possibility that cell
adhesion to Cyr61 may not be mediated through a direct interaction
between Cyr61 and cell surface receptor(s). To exclude this
possibility, we examined the adhesion of HUVECs to purified Cyr61 under
serum-free conditions. In this study, to obliterate the possibility of
serum protein contamination, Cyr61 was purified from serum-free
conditioned medium harvested from Sf9 insect cells passaged only
in serum-free medium. In addition, HUVECs were harvested in 2 mM EDTA, washed, and resuspended in F12K serum-free medium
containing 7 mg/ml BSA. Under these conditions, the adhesion of HUVECs
to Cyr61 was concentration-dependent and saturable (Fig.
2A). Furthermore, cell
adhesion was inhibited by affinity-purified anti-Cyr61 antibodies in a
dose-dependent manner, indicating that the adhesion is an
intrinsic activity of Cyr61 (Fig. 2B). In contrast,
anti-Cyr61 antibodies had no effect on HUVEC adhesion to
vitronectin.
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Cyr61 Interacts with Purified Integrin
v
3--
The observation that RGDS and
LM609 block HUVEC adhesion to Cyr61 suggests that integrin
v
3 may act as a cell surface receptor for
Cyr61. To assess this possibility, we sought to determine whether Cyr61
interacts with
v
3 in purified systems.
Accordingly, we examined direct binding of
v
3 to an affinity matrix onto which Cyr61
was coupled. As controls, vitronectin- or glycine-coupled matrices were
used. In these experiments, purified
v
3
was incubated with the affinity matrices and washed extensively, and
bound integrin was extracted and analyzed by immunoblotting with
anti-
3 antibodies. As shown in Fig.
6,
v
3 bound
to both Cyr61- or vitronectin-coupled Sepharose but not to the control
glycine-coupled matrix. As expected, preincubation of the affinity
matrices with anti-vitronectin antibodies blocked
v
3 binding to vitronectin but not to
Cyr61. Likewise, anti-Cyr61 antibodies blocked
v
3 binding to Cyr61 but not to vitronectin. These results demonstrated a specific interaction between
integrin
v
3 and Cyr61.
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DISCUSSION |
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Cyr61, encoded by a growth factor-inducible immediate-early gene,
belongs to an emerging family of extracellular signaling proteins in
which members (e.g. Cyr61 and Fisp12) have been shown to
promote cell adhesion, migration, and proliferation (5, 7). Sequence
analysis of Cyr61 and other members of this protein family revealed
four conserved domains sharing homology with 1) insulin-like growth
factor-binding proteins, 2) von Willebrand Factor type C domain, 3)
heparin-binding proteins, and 4) the C-terminal domains of some types
of collagens and mucins (30). To date, a cell surface receptor for
these proteins has not been identified. In this study, we investigated
the mechanism by which Cyr61 mediates HUVEC adhesion and identified
integrin v
3 as a cell surface receptor
for Cyr61. This conclusion is based on both functional and biochemical
evidence: 1) HUVEC adhesion to Cyr61 was inhibited by EDTA, an
RGD-containing peptide, and the anti-
v
3
monoclonal antibody LM609; and 2) purified integrin
v
3 bound specifically to an Cyr61-coupled
affinity matrix, and conversely, purified Cyr61 interacted directly
with immobilized
v
3 in a solid phase
receptor binding assay.
Integrin v
3 binds to a broad spectrum of
both RGD-containing and non-RGD-containing ligands (26, 31, 32). The
primary sequence of Cyr61 does not contain the RGD recognition motif
present in
v
3 ligands such as
vitronectin, fibronectin, fibrinogen, thrombospondin, and osteopontin
(31). In addition to Cyr61, at least two other
v
3 ligands also do not contain an RGD
sequence: a cell surface molecule from the immunoglobulin superfamily
CD31/PECAM-1 (32) and metalloproteinase MMP-2 (26). However, RGD
peptides block the interaction between integrin
v
3 and these proteins. Similar inhibition
of Cyr61 binding to
v
3 by RGDS was also
observed in the present study. The inhibitory effect of RGD peptides
may be due to conformational changes of
v
3 upon RGD occupancy, thus masking other
binding sites in the receptor for these non-RGD-containing proteins.
The ability of Cyr61 to mediate HUVEC adhesion was observed under both
serum-free and serum-containing conditions (Figs. 2-5). Within a short
adhesion period (15 min), vitronectin was capable of inhibiting HUVEC
adhesion to Cyr61, consistent with competition of these proteins for
binding to the same receptor. However, inhibition was not observed when
cell adhesion was allowed to proceed for a longer period (30 min) (Fig.
4). This is likely due to the nonequilibrium nature of cell adhesion to
immobilized substrates mediated through v
3 (28). The observation that HUVECs
adhered to Cyr61 via
v
3 when challenged
with 50 µg/ml vitronectin (Fig. 4) or 10% serum (Fig. 5) indicate
that Cyr61 may function as a physiological adhesion substrate even in
the presence of RGD-containing
v
3 ligands.
Although it is currently unclear how Cyr61 interacts with
v
3, studies employing phage display
libraries have revealed sequences other than RGD capable of binding to
the integrins
v
3 and
5
1 (33-35). Furthermore, data from
studies on disintegrins and phage display libraries indicate that
certain peptide sequences bind integrins with substantially higher
affinity when cyclized via disulfide bonding (33-39). In this regard,
the processed, secreted Cyr61 contains 38 cysteine residues, thereby
providing the possibility of creating cyclized sequences that interact
with
v
3. Identification of the
recognition sequence within Cyr61 would be essential for our
understanding of the ligand diversity of the
v
3 integrin, as well as the
structure-function relationship of the newly discovered Cyr61 family of
growth-related proteins.
The inducible expression of Cyr61 by growth factors suggests that Cyr61
may function as a transient and localized signaling molecule regulating
biological processes in which cell adhesion plays a role. Cell
migration, proliferation, and differentiation are examples of such
processes. Although we have established that the cell adhesive
properties of Cyr61 are mediated through interaction with integrin
v
3, whether other activities of Cyr61 are
also manifested through the same receptor is not yet known. It is
tempting, however, to speculate that the chemotactic and proliferative
activities of Cyr61 may also be mediated through the
v
3 integrin. Although these processes
involve much more complex cellular responses than adhesion, they are
completely consistent with the capabilities of integrins to induce
these signaling events. In this regard, it is widely accepted that cell
adhesion receptors are crucial for cell migration (10, 15), and the
regulation of integrin affinity to proteins in the ECM is likely a
driving force of cell migration (40, 41). Therefore, the modulation of
the affinity of integrin
v
3 toward Cyr61
and other ligands in the ECM may account for the chemotactic activity
of Cyr61. The participation of integrins in modulation of growth
factor-mediated signaling is also well established (16, 17). For
example, regulation of cell cycle progression by
1 integrins
(42-44) and integrin
v
3 (45, 46) have
been reported. Consistent with the observation that Cyr61 is able to
augment basic fibroblast growth factor-induced and platelet-derived
growth factor-induced DNA synthesis (5), integrin- and growth
factor-induced signaling cascades have been shown to intersect and
converge (16, 17, 47).
The finding that Cyr61 interacts with integrin
v
3 also provides insights into its
possible biological functions. Although Cyr61 is a secreted protein, it
is tightly associated with the ECM and the cell surface upon secretion
and is thus likely to act locally in either an autocrine or a paracrine
fashion (4). During development, Cyr61 is highly expressed in the
placenta, most notably in the trophoblastic giant cells and
trophoblasts of the ectoplacental cone (8).
v
3 is the only integrin found to be
expressed on the apical surface of trophoblasts and on the uterine
epithelium during the period of receptivity for implantation (48, 49),
and
v
3 continues to be expressed in the
trophoblastic giant cells and ectoplacental cone in later stages (49,
50). Because Cyr61 is likely to be deposited from the trophectoderm onto the neighboring matrix, it can be hypothesized that interaction between Cyr61 and
v
3 may help to mediate
attachment of the embyro to the endometrium through cell-matrix
interactions. Furthermore, in the post-implantation embryo, Cyr61
interaction with
v
3 may mediate
attachment to the decidua. In addition, because Cyr61 has been shown to
promote chondrogenesis to form the embryonic skeleton (9), the presence
of both Cyr61 (8, 9) and
v
3 (51) in
chondrocytes suggests that their interaction may play a role in this
process. In the circulatory system, Cyr61 and
v
3 are coexpressed in vascular smooth
muscle cells (8, 52) and in microvascular endothelial cells
(53).2 In HUVECs, we
previously demonstrated that Cyr61 mediates cell adhesion and augments
basic fibroblast growth factor-induced DNA synthesis (5, 54). Because
basic fibroblast growth factor, which induces angiogenesis, activates
the synthesis of both Cyr61 (3, 55) and
v
3 (53), these observations suggest that Cyr61 may be involved in angiogenesis during wound repair. In this
regard,
v
3 has been shown to mediate
angiogenesis in chicken chorioallantoic membrane (56) and basic
fibroblast growth factor-induced neovascularization in rabbit cornea
(57). Our finding that Cyr61 is a ligand for
v
3 is consistent with its potential role
in growth factor-induced angiogenesis.
In sum, we demonstrated that integrin v
3
serves as an adhesion receptor for Cyr61 on HUVECs. Although we cannot
exclude the possibility that Cyr61 may also bind to other cell surface molecules, such as heparan sulfate proteoglycans and growth factor receptors, the interaction of Cyr61 with integrin
v
3 may account for most, if not all, of
the ascribed physiological activities of Cyr61 to date. Thus, Cyr61 and
other members of its family may represent a novel class of signaling
molecules, providing additional functional links between the growth
factor-dependent and matrix-dependent signaling
events.
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ACKNOWLEDGEMENTS |
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We thank Dr. D. A. Cheresh for a generous gift of monoclonal antibody LM609, C. C. Chen and D. G. Pestov for expert help, and our colleagues for helpful discussions.
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FOOTNOTES |
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* This work was in part supported by National Institutes of Health Grants CA46565 (to L. F. L.) and HL41793 (to S. C.-T. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Present address: Laboratory of Molecular Mechanisms of Transcription, NCI, NIH, Frederick, MD 21702.
Supported by an Established Investigator Award from the
American Heart Association and Genetech.
** Supported by an Established Investigator Award from the American Heart Association. To whom correspondence should be addressed: Department of Molecular Genetics (M/C 669), University of Illinois at Chicago, 900 South Ashland Ave., Chicago, IL 60607-7170. Tel.: 312-996-6978; Fax: 312-996-7034.
1 The abbreviations used are: ECM, extracellular matrix; HUVEC, human umbilical vein endothelial cell; PBS, phosphate-buffered saline; BSA, bovine serum albumin.
2 C. C. Chen and L. F. Lau, unpublished observations.
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REFERENCES |
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