CYR61 Stimulates Human Skin Fibroblast Migration through Integrin
v
5 and Enhances Mitogenesis through
Integrin
v
3, Independent of Its
Carboxyl-terminal Domain*
Tatiana M.
Grzeszkiewicz
,
Deborah J.
Kirschling§,
Ningyu
Chen
, and
Lester F.
Lau
¶
From the
Department of Molecular Genetics,
University of Illinois College of Medicine,
Chicago, Illinois 60607 and § Munin Corporation,
Chicago, Illinois 60612
Received for publication, February 1, 2001, and in revised form, March 26, 2001
 |
ABSTRACT |
CYR61, an angiogenic factor and
a member of the CCN protein family, is an extracellular
matrix-associated, heparin-binding protein that mediates cell
adhesion, promotes cell migration, and enhances growth
factor-stimulated cell proliferation. CYR61 induces angiogenesis and
promotes tumor growth in vivo and is expressed in dermal
fibroblasts during cutaneous wound healing. It has been demonstrated
recently that adhesion of primary skin fibroblasts to CYR61 is mediated
through integrin
6
1 and cell surface
heparan sulfate proteoglycans, resulting in adhesive signaling and
up-regulation of matrix metalloproteinases 1 and 3. CYR61 is composed
of four discrete structural domains that bear sequence similarities to
the insulin-like growth factor-binding proteins, von Willebrand factor
type C repeat, thrombospondin type 1 repeat, and a carboxyl-terminal
(CT) domain that resembles cysteine knots found in some growth factors.
In this study, we show that a CYR61 mutant (CYR61
CT) that has the CT
domain deleted is unable to support adhesion of primary human skin
fibroblasts but is still able to stimulate chemotaxis and enhance basic
fibroblast growth factor-induced mitogenesis similar to wild type. In
addition, fibroblast migration to CYR61 is mediated through integrin
v
5 but not integrins
6
1 or
v
3.
Furthermore, we show that CYR61 binds directly to purified integrin
v
5 in vitro. By contrast, CYR61 enhancement of basic fibroblast growth factor-induced DNA synthesis is mediated through integrin
v
3, a known receptor for CYR61 that
mediates CYR61-dependent cell adhesion and chemotaxis in
vascular endothelial cells. Thus, CYR61 promotes primary human fibroblast adhesion, migration, and mitogenesis through integrins
6
1,
v
5, and
v
3, respectively. Together, these
findings establish CYR61 as a novel ligand for integrin
v
5 and show that CYR61 interacts with
distinct integrins to mediate disparate activities in a cell
type-specific manner.
 |
INTRODUCTION |
CYR61, an angiogenic inducer, is a secreted,
ECM1-associated protein and a
member of the CCN family, which also consists of CTGF, nephroblastoma
overexpressed, Elm-1/WISP-1, Cop-1/WISP-2, and WISP-3 (1, 2). A
striking feature of the CCN protein family is their organization into
four conserved modular domains that share sequence similarities with
insulin-like growth factor-binding proteins, von Willebrand factor type
C repeat, thrombospondin type 1 repeat, and growth factor cysteine
knots (Fig. 1) (3). CCN family members contain all four conserved
domains with the exception of WISP-2, which lacks precisely the
carboxyl-terminal (CT) domain (4, 5). Since each domain is encoded by a
separate exon, the CCN gene family probably arose through exon
shuffling, and the overall functions of CCN proteins may be programmed
by the combinatorial actions of each domain acting independently as
well as interdependently.
The activities of CYR61 are both cell type- and context-specific. In
fibroblasts and endothelial cells, CYR61 mediates cell adhesion,
stimulates cell migration, and potentiates growth factor-induced DNA
synthesis (6, 7). In micromass cultures of mouse limb bud mesenchymal
cells, CYR61 accelerates and promotes chondrogenic differentiation (8).
CYR61 induces angiogenesis in vivo and enhances the
tumorigenicity of human tumor cells in immunodeficient mice by
increasing tumor size and vascularization (9). CYR61 also up-regulates
expression of matrix metalloproteinases 1 and 3 (10), enzymes that can
promote matrix remodeling in processes such as angiogenesis and wound
healing. These demonstrated activities of CYR61 are consistent with its
expression in chondrogenic and angiogenic cell types during development
(11, 12) and in granulation tissue during cutaneous wound healing
(7).
Integrin receptors mediate at least some of the activities of CYR61 in
various cell types. Integrins are heterodimeric cell surface receptors
that are functionally versatile and capable of regulating cell
adhesion, migration, proliferation, differentiation, and survival (13,
14). Recombinant CYR61 protein is a ligand of, and binds directly to,
integrins
v
3 and
IIb
3. Interaction between these integrins
and CYR61 mediates endothelial cell adhesion and migration (integrin
v
3) or blood platelet adhesion (integrin
IIb
3) (9, 15, 16). CYR61 also supports
the adhesion of primary human fibroblasts through integrin
6
1 and cell surface heparan sulfate
proteoglycans (17), resulting in adhesive signaling manifested by
persistent formation of filopodia and lamellipodia, formation of
integrin subunits
6- and
1-containing
focal complexes, activation of focal adhesion kinase and
mitogen-activated protein kinases, and up-regulation of matrix
metalloproteinases 1 and 3 (10).
The organization of CYR61 and CCN proteins into four discrete
structural domains that are homologous to disparate families of
proteins suggests that these domains might function both independently and in concert. The domains of CCN proteins are separated by
protease-sensitive residues, raising the possibility that distinct
forms of the proteins can be generated by proteolysis. It has been
reported that another family member, CTGF, is proteolytically processed
such that a mitogenically active fragment corresponding to the CT
domain alone can be isolated from porcine uterine fluids (18). To
investigate the structure-function relationship of CCN proteins, we
have constructed a mutant form of CYR61 (CYR61
CT) that specifically
lacks the CT domain, and we examined the functional consequences of
such a deletion. Furthermore, we sought to identify the cell surface receptors that mediate the chemotactic and mitogenesis enhancing activities of CYR61 in primary human fibroblasts.
In the present study, we show that removal of the CT domain abrogates
the ability of CYR61 to support cell adhesion in primary human
fibroblasts but has no effect on stimulation of cell migration or
enhancement of bFGF-induced DNA synthesis. Furthermore, whereas fibroblast adhesion to CYR61 is mediated through integrin
6
1 and heparan sulfate proteoglycans,
CYR61-dependent chemotaxis and enhancement of DNA synthesis
are mediated through integrins
v
5 and
v
3, respectively. We also show that CYR61
binds directly to purified integrin
v
5 in vitro. These findings
establish CYR61 as a novel ligand for integrin
v
5, identify a functional division within
the modular structure of the protein, and demonstrate that different
integrin receptors mediate distinct activities of CYR61.
 |
MATERIALS AND METHODS |
Antibodies, Peptides, and Reagents--
Function-blocking mAbs
against integrins were purchased from Chemicon, Inc., including
NKI-GoH3 (anti-
6), NKI-M9 and AV1 (anti-
V), LM609 (anti-
V
3),
P1F6 (anti-
V
5), JB1A
(anti-
1), and B3A (anti-
3), as was
purified mouse IgG1. Polyclonal anti-CYR61 antibodies were raised in
rabbits as described previously (19). GRGDSP and GRGESP synthetic
peptides were purchased from Life Technologies, Inc. Echistatin and
heparin (sodium salt, from porcine intestinal mucosa) were from Sigma.
Horseradish peroxidase-conjugated donkey anti-rabbit antibodies were
obtained from Amersham Pharmacia Biotech.
Expression and Purification of CYR61
CT--
A construct
encoding a CYR61 deletion mutant removing the CT domain, CYR61
CT,
was produced by ligation of the coding sequence for the first three
domains to an enterokinase recognition signal and polyhistidine tract
assembled from synthetic oligonucleotides. Polymerase chain reactions
with the human CYR61 cDNA as template were carried out
at 56 °C, 30 cycles, 2 mM MgCl2 using
the primers 5'-CGGGATCCGCCTGTCCGCTGCACACCAGCTTG (corresponding
to nucleotides 51-74) and 5'-CGGAATTCTTTCAGGCTGCTGTACACTGGCTGTC
(nucleotides 943-966), thus yielding the coding sequence for the
IGFBP, VWC, and TSP1 domains while introducing the restriction sites
BglII (5') and EcoRI (3') for cloning purposes.
The histidine tag was assembled as described (20) from the following
oligonucleotides: 5'-GGAATTCGACGATGACGATAAGCATCATCACCAT;
5'-CACCACCATCACAGTGGATAATCTAGAGC; 5'-TGATGCTTATCGTCATCGTCGAATTCC; and
5'-GCTCTAGAATTTCCACTGTGATGGTGGTGATGGTGA containing a stop codon
and the restriction sites EcoRI (5') and XbaI
(3'). The polymerase chain reaction product and assembled histidine-tagged oligonucleotides were purified by agarose gel electrophoresis, digested with EcoRI, and ligated. The
resulting product was digested with BglII and
XbaI and cloned into the corresponding sites in the
baculovirus expression vector pBluebac 4.5 (Invitrogen). The sequence
and orientation of the CYR61
CT construct was confirmed by DNA sequencing.
A recombinant baculovirus stock was generated as described
previously (6). Recombinant CYR61
CT protein was produced in a
serum-free baculovirus expression system using High Five insect cells
essentially as described (6) with modifications. Cells were maintained
under serum-free conditions in EX-CELL 400 medium (JRH Biosciences),
infected at a multiplicity of infection of 10, and collected 40 h
post-infection. Because the conditioned medium contains chelating
agents capable of stripping the nickel-agarose column, it was first
adjusted to 50 mM sodium phosphate, precipitated with 50%
(w/v) ammonium sulfate, and centrifuged at 5500 × g
for 30 min. The resulting pellet was resuspended in and dialyzed
against native binding buffer (20 mM
Na2HPO4, 0.8 NaCl, 0.5 mM PMSF, pH 7.2) for 8 h and then applied in batches to a nickel-agarose
column (Qiagen). The column was washed with native binding buffer, low stringency buffer (20 mM Na2HPO4,
0.8 NaCl, 0.5 mM PMSF, pH 6.0), and high stringency buffer
(20 mM Na2HPO4, 0.8 NaCl, 0.5 mM PMSF, pH 5.5), before being eluted in 20 mM
Na2HPO4, 0.8 NaCl, 0.5 mM PMSF, pH
4.5, and neutralized with HEPES (40 mM). Remaining proteins were removed from the column using EDTA stripping buffer (0.5 M EDTA, pH 8.0). The resulting product was analyzed for
purity and concentration using SDS-polyacrylamide gel electrophoresis followed by Coomassie Blue staining and immunoblotting.
CYR61, CYR61DM, and Matrix Proteins--
Recombinant CYR61DM was
produced in a serum-free baculovirus system using Sf9 cells as
described previously (17). For an enterokinase recognition
signal/polyhistidine tract tagged version of human CYR61
recombinant protein, the synthesized oligonucleotides 5'-CCCGGAATTCAGGGACGATGACGATAAGCATCACCATCAC and
5'-CCCCCCAAGCTTAGTGATGGTGATGGTGATGCTTATCGTC were annealed, filled
using Pfu DNA polymerase, double digested with
EcoRI and HindIII, and ligated into the
corresponding sites of the pBluebac4.5 plasmid (Invitrogen). Human
CYR61 cDNA was digested sequentially with
ApoI and XbaI, gel-purified, and ligated into the
XbaI and EcoRI sites of the engineered
pBluebac4.5 vector. A recombinant baculovirus stock was generated as
reported previously (6) and produced as described for CYR61
CT.
Recombinant CYR61 protein was purified by Sepharose S column
chromatography as described previously (6), and pooled CYR61 fractions
were subjected to nickel column chromatography as detailed for
CYR61
CT. Recombinant protein purity and concentration were assayed
using polyacrylamide gel electrophoresis followed by Coomassie Blue
staining and immunoblotting. Human vitronectin, fibronectin,
recombinant platelet-derived growth factor, and recombinant bFGF were
purchased from Life Technologies, Inc. Integrin
V
5 purified via mAb
affinity-chromatography was obtained from Chemicon, Inc.
Cell Culture and Adhesion Assay--
Primary human foreskin
fibroblasts 1064SK (ATCC CRL-2076, passage 2) were kept in Iscove's
modified Dulbecco's medium (Life Technologies, Inc.) with 10% fetal
bovine serum (Intergen). Adhesion assays were conducted as described
previously (15). Briefly, fibroblasts were harvested in
phosphate-buffered saline (137 mM NaCl, 2.7 mM
KCl, 4.3 mM Na2HPO4, pH 7.3) with
2.5 mM EDTA, resuspended in serum-free Iscove's modified
Dulbecco's medium containing 0.5% BSA to 5 × 105
cells/ml. To each well, 50 µl of cell suspension was plated and allowed to adhere for 30 min at 37 °C. After incubation, cells were
rinsed once each with plating medium and phosphate-buffered saline, and
adherent cells were fixed with 10% formalin, stained with methylene
blue, and quantified by dye extraction with measurement of absorbance
at 620 nm.
Cell Migration Assay--
A modified Boyden chamber was used to
determine 1064SK fibroblast migration as described previously (21).
Test proteins were diluted in Iscove's modified Dulbecco's medium
containing 0.5% BSA and loaded in the lower well of the chamber in
quadruplicate. The lower well was then covered with a polycarbonate
filter (5 µm pore diameter, Nucleopore), which was treated
with 2.9% (v/v) glacial acetic acid overnight, rinsed three times in
distilled water for 1 h, and coated with 0.01% gelatin (Difco)
for 2 h. Fibroblasts were trypsinized, resuspended in serum-free
Iscove's modified Dulbecco's medium containing 0.5% BSA, and applied
to the upper wells (5 × 104 cells/well). After a 6-h
incubation at 37 °C, 5% CO2, the membrane was removed
and stained with Diff-Quik (Dade-Behring). The chemotactic response was
determined by counting the total number of cells migrated in 10 randomly selected microscope fields at × 400 magnification.
Thymidine Incorporation Assay--
DNA synthesis was measured as
described previously (6) with minor modifications. 1064SK fibroblasts
were plated on 24-well plates at 1 × 104 cells/well
and grown in Iscove's modified Dulbecco's medium with 10% fetal
bovine serum for 48 h, rinsed with phosphate-buffered saline, and
incubated with serum-free medium containing 0.5% BSA for an additional
48 h. Where indicated, peptides or antibodies were diluted in
serum-free medium and incubated with cells for 1 h at room
temperature prior to their removal and the addition of assayed
proteins. Fresh serum-free medium containing designated proteins and 1 µCi/well [3H]thymidine were simultaneously added, and
after incubation for 21 h, cells were washed with
phosphate-buffered saline and fixed with 10% trichloroacetic acid. DNA
was dissolved in 0.1 N NaOH and incorporated thymidine
measured using a scintillation counter.
Solid Phase Binding Assay--
Binding of CYR61 to immobilized
integrin
V
5 was detected via ELISA
adapted from a study of CYR61 binding to integrin
V
3 (15). Microtiter wells were coated
with 50 µl/well of integrin
V
5 (1 µg/ml) or BSA (1 µg/ml) at room temperature for 1 h followed by incubation at 4 °C for 36 h. Plates were blocked with 0.5% gelatin (Bio-Rad) for 6 h at 37 °C. Plates were rinsed four
times with phosphate-buffered saline containing divalent cations (1 mM CaCl2, 1 mM MgCl2,
pH 7.5) after blocking, and incubated with serially diluted CYR61
protein (50 µl/well) in blocking solution (0.5% gelatin, 137 mM NaCl, 2.7 mM KCl, 4.3 mM
Na2HPO4, 1 mM CaCl2, 1 mM MgCl2, pH 7.5) for 3 h at room
temperature. For detection of bound CYR61, plates were incubated for
2 h with polyclonal anti-CYR61 (1:2500) followed by horseradish
peroxidase-conjugated secondary antibody (1:1000) for 1 h. All
incubations were performed at 37 °C, and plates were rinsed four
times with phosphate-buffered saline between steps and six times
following secondary antibody incubation. The color reaction was
developed using a horseradish peroxidase immunoassay kit
(Zymed Laboratories Inc.), and absorbance was measured
at 420 nm. Quantitation of CYR61 coated on microtiter wells (Fig.
3B) was conducted using ELISA essentially as described above, except microtiter wells were coated with indicated proteins at
10 µg/ml. Plates were then blocked, rinsed, and probed with anti-CYR61 antibodies as detailed.
 |
RESULTS |
The CYR61 CT domain is indispensable for supporting human skin
fibroblast adhesion. To dissect the structural requirements for the
various activities of CYR61, we have created CYR61 mutants that either
has deleted precisely the CT domain (CYR61
CT) or harbors amino acid
substitutions (Cyr61DM) in this domain destroying the heparin-binding
sites (Fig. 1). Since the CT domain
contains the heparin-binding sites, we anticipated that deleting this
domain would render the protein unable to bind heparin or Sepharose S columns. Therefore, CYR61
CT has been histidine-tagged for the purpose of purification. A similarly tagged version of the human wild
type CYR61 protein was produced and found to be functionally equivalent
to the previously characterized, non-tagged CYR61 protein (data not
shown). Both recombinant CYR61 and CYR61
CT proteins were produced in
the baculovirus system and purified using the histidine tag via
nickel-agarose column chromatography (22). The histidine-tagged
CYR61
CT bound well to the nickel column, allowing its purification
to near homogeneity as judged by SDS-polyacrylamide gel electrophoresis
(Fig. 2A) and immunoblotting
with affinity-purified anti-CYR61 antibodies (Fig. 2B).

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Fig. 1.
Schematic representation of CYR61 protein
constructs. Illustrated are the secretory signal (SP)
and four modular domains (insulin-like growth factor binding protein
(IGFBP); von Willebrand factor type C repeat
(VWC); thrombospondin type 1 repeat (TSP1); and
carboxyl-terminal domain (CT)). Where indicated, a
polyhistidine tract (HIS) has been added to the carboxyl
terminus of the protein, or site-directed mutagenesis was used to
replace the lysine and arginine residues in the heparin-binding motifs
(HBS) with alanine.
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Fig. 2.
CYR61 CT protein
purification. A, 30 µl of nickel-agarose-purified
CYR61 CT eluate was loaded per lane and electrophoresed on an
SDS-polyacrylamide gel, followed by staining with Coomassie Brilliant
Blue. 1-12, fraction number; MW, molecular
weight marker. B, 1 µl of nickel-agarose-purified
CYR61 CT protein eluate was loaded per lane and electrophoresed on an
SDS-polyacrylamide gel, followed by immunoblotting with anti-CYR61
antibodies. 1-12, fraction number; CM,
conditioned medium.
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To analyze the functional capabilities of CYR61
CT, we first
addressed its ability to support the adhesion of 1064SK primary human
skin fibroblasts. Test proteins were coated onto microtiter wells, and
cells were allowed to attach under serum-free conditions. Wild type
CYR61 supported fibroblast adhesion in a dose-dependent manner, reaching maximal adhesion when the coating concentration was 2 µg/ml (Fig. 3A), consistent
with previous studies (17). CYR61
CT was completely unable to mediate
cell adhesion, even at a coating concentration as high as 100 µg/ml.
This adhesion defect was not due to the inability of CYR61
CT to coat
the plastic microtiter wells. When wells were coated with either wild
type CYR61 or CYR61
CT, both bound proteins could be readily detected to similar levels with anti-CYR61 polyclonal antibodies by ELISA (Fig.
3B). These results indicate that the CT domain is
indispensable for fibroblast adhesion. This observation is consistent
with the finding that Cyr61DM, with the heparin-binding sites present
in the CT domain mutated, is unable to support fibroblast adhesion (17).

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Fig. 3.
CYR61 CT does not
support fibroblast adhesion. A, cell adhesion assays
were performed with washed 1064SK fibroblasts detached with 2.5 mM EDTA and resuspended in serum-free Iscove's modified
Dulbecco's medium at 5 × 105 cells/ml. 50 µl of
cell suspension was plated on each microtiter well coated with the
indicated amount of protein. After incubation at 37 °C for 30 min,
adherent cells were fixed and stained with methylene blue, and
extracted dye was quantified by absorbance at 620 nm. B,
microtiter wells were coated with 10 µg/ml indicated proteins. After
blocking with 0.5% gelatin, proteins were detected using an ELISA
system with polyclonal anti-CYR61 antibodies. Data shown for both
panels are mean ± S.D. of triplicate determinations and are
representative of three experiments.
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|
Integrin
v
5 Mediates Human Skin
Fibroblast Migration to CYR61 and CYR61
CT--
We have previously
found that CYR61 promotes migration of both fibroblasts and endothelial
cells and that endothelial cell chemotaxis to CYR61 is mediated through
integrin
v
3 (6, 9). By using a modified
Boyden chamber assay, we investigated whether the CT domain was
required for fibroblast migration. As shown in Fig.
4A, 1064SK fibroblasts
migrated to soluble wild type CYR61 in a dose-dependent
manner with maximal migration occurring at 2 µg/ml. Likewise,
fibroblasts migrated to CYR61
CT and Cyr61DM with similar dose
responses (Fig. 4, B and C). To investigate the
possible role of the heparin binding activity of CYR61, we co-incubated
fibroblasts with soluble heparin, a condition under which fibroblast
adhesion to CYR61 could be abolished due to saturation of available
heparin-binding sites on CYR61 (17). Since the CYR61
CT lacking the
heparin-binding motifs still induced migration, it was not surprising
that heparin had no effect on fibroblast migration to either the wild
type or mutant protein (Fig.
5A). Therefore, the CT domain
of CYR61 and its heparin-binding capability are not required for
CYR61-stimulated fibroblast migration.

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Fig. 4.
The CT domain is dispensable for fibroblast
migration. A modified Boyden chamber was used to measure
fibroblast migration. 1064SK fibroblasts were detached with trypsin and
resuspended in serum-free Iscove's modified Dulbecco's medium, and
5 × 104 cells were loaded per well. Cells were
allowed to migrate for 6 h at 37 °C before being fixed and
stained. Cells placed in the upper chamber that migrated to the lower
chamber were counted in 10 random high power fields (HPF).
Serially diluted proteins at indicated concentrations were used as the
chemoattractant. A, migration to CYR61. B,
migration to CYR61 CT. C, migration to CYR61DM. Data shown
for all panels are mean ± S.D. of quadruplicate determinations
and are representative of three experiments.
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Fig. 5.
Migration to CYR61 protein is not mediated by
integrin
6 1
or heparan sulfate proteoglycans. Migration assays were performed
using 1064SK fibroblasts in a modified Boyden chamber. A,
cells were treated with heparin (10 µg/ml) or anti- 6
mAb clone GoH3 (50 µg/ml) for 1 h prior to chamber loading and
exposure to BSA (0.5%), platelet-derived growth factor (10 ng/ml),
CYR61 (2 µg/ml), or CYR61 CT (2 µg/ml). B, cells were
treated with anti- 1 mAb clone JB1A (50 µg/ml) for
1 h prior to chamber loading and exposure to BSA (0.5%), CYR61 (2 µg/ml), CYR61 CT (2 µg/ml), fibronectin (FN)
(10 ng/ml), or vitronectin (VN) (10 µg/ml). Data shown for
all panels are mean ± S.D. of quadruplicate determinations and
are representative of three experiments.
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Since fibroblast adhesion to CYR61 is mediated through integrin
6
1 and cell surface heparan sulfate
proteoglycans function as co-receptors (17), we investigated whether
integrin
6
1 was responsible for
CYR61-stimulated fibroblast migration. Cells were preincubated with
either a mAb against integrin
6 (GoH3) or integrin
1 (JB1A) for 1 h prior to cell migration assay.
Neither mAb had any effect on cell migration to either CYR61 or
CYR61
CT (Fig. 5). Additionally, fibroblast migration to CYR61 was
not blocked even when cells were treated with both heparin and GoH3 simultaneously (data not shown). By contrast, cell migration to fibronectin (which binds integrin
5
1),
but not vitronectin (which binds integrins
v
3 and
v
5),
was completely blocked by mAb JB1A directed against the integrin
1 subunit. These results indicate that unlike fibroblast
adhesion, fibroblast migration to CYR61 is not mediated through
integrin
6
1.
To elucidate the mechanism responsible for CYR61-stimulated fibroblast
migration, we next focused on the
v integrins since CYR61 has been demonstrated to be a ligand of integrin
v
3 (15). By using a mAb (NKI-M9) against
the integrin
v subunit, we were able to inhibit CYR61-
and CYR61
CT-stimulated migration (Fig. 6A), thus confirming the
involvement of an
v integrin. As expected, NKI-M9
inhibited cell migration to vitronectin but not to fibronectin. LM609,
a mAb with specificity for the integrin
v
3 heterodimer (23), had no effect on
cell migration to CYR61 but blocked migration to vitronectin as
expected (Fig. 6B). Therefore, in contrast to migration in
endothelial cells (9), CYR61-stimulated fibroblast migration is not
mediated through integrin
v
3. Since
integrin
v
5 is expressed in fibroblasts
and has been shown to mediate activation-dependent cell
migration (24-27), we examined its ability to mediate CYR61-induced
migration. When fibroblasts were treated with P1F6, a mAb against
integrin
v
5, migration to both CYR61 and
CYR61
CT protein was blocked completely (Fig. 6C). As
expected, P1F6 had no effect on migration to fibronectin. Together,
these results show that fibroblast migration to CYR61 is mediated
through integrin
v
5.

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Fig. 6.
Integrin
v 5
mediates migration to CYR61 and CYR61 CT
protein. Migration assays were performed in a modified Boyden
chamber using 1064SK fibroblasts. Cells were treated for 1 h with
specified mAbs (50 µg/ml) before exposure to BSA (0.5%), CYR61 (2 µg/ml), CYR61 CT (2 µg/ml), fibronectin (FN)
(10 ng/ml), or vitronectin (VN) (10 µg/ml). A,
anti- v mAb clone NKI-M9. B,
anti- v 3 mAb clone LM609. C,
anti- v 5 mAb clone P1F6. Data shown for
all panels are mean ± S.D. of quadruplicate determinations and
are representative of three experiments.
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CYR61 Binds Directly to Integrin
v
5
in Vitro--
We have shown previously that CYR61 is a ligand of, and
binds directly to, integrins
v
3 and
IIb
3 (15, 16). That CYR61-stimulated fibroblast migration is mediated through integrin
v
5 prompted us to investigate whether
CYR61 can bind directly to this integrin. In a solid phase binding
assay, purified integrin
v
5 receptor was
immobilized on microtiter wells onto which CYR61 was allowed to bind.
The reaction was detected by ELISA using anti-CYR61 antibodies. Fig.
7A shows that CYR61 bound to
immobilized integrin
v
5 in a
dose-dependent and saturable manner, with half-saturation
occurring at ~6 nM (0.25 µg/ml) CYR61. As expected,
there was no significant interaction between CYR61 and the BSA
control.

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Fig. 7.
CYR61 binds directly to immobilized
integrin
v 5.
Microtiter wells were coated with purified
v 5 receptor or BSA (1 µg/ml each) as
indicated. A, after gelatin blocking, varying concentrations
of CYR61 were added and binding proceeded at room temperature for
3 h. Bound CYR61 was detected using ELISA. B, plates
were treated for 1 h prior to CYR61 incubation with EDTA (5 mM), EDTA + Mg2+ (10 mM), GRGDSP
peptide (0.2 mM), GRGESP peptide (0.2 mM),
anti- v 5 mAb clone P1F6 (20 µg/ml), and
anti- v 3 mAb clone LM609 (20 µg/ml).
Data shown for all panels are mean ± S.D. of triplicate
determinations and are representative of three experiments.
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To determine the specificity of the interaction between CYR61 and
integrin
v
5, inhibitors of integrin
v
5 function were used as shown in Fig.
7B. The binding of CYR61 to immobilized
v
5 was blocked by 5 mM EDTA
and restored by the addition of 10 mM Mg2+,
consistent with the divalent cation requirement of integrin
v
5 (28, 29). Since integrin
v
5 recognizes protein ligands with the
RGD sequence motif and is inhibited by soluble RGD peptides (30), we
examined the effect of RGD-containing peptides. The peptide GRGDSP at
0.2 mM, but not the control peptide GRGESP, completely
abrogated CYR61 binding to integrin
v
5. A
function-blocking mAb against integrin
v
5
(P1F6) inhibited CYR61 binding completely, whereas the
anti-
v
3 mAb (LM609) exhibited only a
partial inhibitory effect. Taken together, these results show that
CYR61 can bind directly to purified integrin
V
5 in vitro, consistent with
functional studies showing that CYR61 stimulates fibroblast migration
through this integrin (Fig. 7).
CYR61 and CYR61
CT Enhance bFGF-induced DNA Synthesis
in Fibroblasts through Integrin
v
3--
Although CYR61 is not mitogenic
alone, its ability to enhance growth factor-induced DNA synthesis has
been established for fibroblasts and endothelial cells (6, 7). Thus, we
were interested in elucidating what activity CYR61
CT may have on
1064SK fibroblast proliferation. When added as a soluble factor to
fibroblasts under serum-free conditions, CYR61
CT alone did not
elicit a mitogenic response (Fig.
8A) as described previously
for wild type CYR61 (6). However, when added in the presence of a
suboptimal concentration of bFGF (10 ng/ml), CYR61
CT elicited a
2-fold increase over bFGF treatment in thymidine incorporation similar
to wild type CYR61 (Fig. 8A). Furthermore, the
mitogenesis-enhancing effect of CYR61
CT is
dose-dependent and saturable, with maximal potentiation
occurring at 3 µg/ml under serum-free conditions (Fig.
8B). These studies show that the CT domain is not required
for CYR61 to enhance growth factor-induced DNA synthesis.

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 8.
CYR61 CT enhances
bFGF-induced DNA synthesis in 1064SK fibroblasts. The effect of
soluble CYR61 CT on bFGF-induced mitogenesis under serum-free
conditions was assessed on fibroblasts attached to 24-well plates.
A, serum-starved cells were treated with BSA (0.5%),
CYR61 CT (3 µg/ml), bFGF (10 ng/ml), or CYR61 (3 µg/ml) and
[3H]thymidine for 21 h before incorporation was
measured. B, serum-starved cells were treated with 10 ng/ml
bFGF, the indicated concentration of CYR61 CT, and
[3H]thymidine for 21 h before incorporation was
measured. Data shown for all panels are mean ± S.D. of triplicate
determinations and are representative of three experiments.
|
|
Since integrin receptors have been shown to mediate many of the
activities of CYR61, we hypothesized that they may also be involved in
this proliferation-enhancing effect. To investigate this possibility,
we first treated cells with GRGDSP or GRGESP peptide for 1 h
before the addition of CYR61 and bFGF. Interestingly, the peptide
GRGDSP at 0.2 mM, but not the control peptide GRGESP, completely neutralized the CYR61 enhancement of bFGF-induced 1064SK fibroblast mitogenesis (Fig.
9A). By contrast, DNA
synthesis induced by bFGF alone was unaffected. This result indicated
that an RGD-sensitive integrin may be responsible for mediating the
proliferation-enhancing effect of CYR61. Furthermore, when cells were
treated with echistatin, a disintegrin with binding preference for
3 integrins (31), CYR61 enhancement of bFGF-induced
proliferation was similarly abrogated completely. These results,
together with the fact that CYR61 is a known ligand of integrin
v
3 (15), suggested to us that integrin
v
3 might mediate the
mitogenesis-enhancing effects of CYR61. To investigate this possibility
further, function-blocking mAbs against relevant integrins were used in
mitogenesis assays. Consistent with the blocking effect of echistatin,
mAbs against integrin
v (AV1) or
3 (B3A)
completely abolished CYR61-enhanced mitogenesis but had no effect on
bFGF-induced mitogenesis (Fig. 9B). However, a mAb against
integrin
v
5 (P1F6) did not have any
effect, indicating that integrin
v
5 does
not play a role in CYR61 enhancement of mitogenesis. By contrast,
LM609, a mAb directed against the integrin
v
3 heterodimer, completely abolished CYR61- or CYR61
CT-enhanced mitogenesis, whereas the control IgG had
no effect (Fig. 9C). Together, these results show that
integrin
v
3, but not integrin
v
5, mediates the enhancement of
bFGF-induced mitogenesis by CYR61. Moreover, CYR61
CT is functionally
equivalent to the wild type protein in this assay, indicating that the
CT domain is not required for CYR61 to stimulate growth factor-induced mitogenesis. These results suggest that a binding site for integrin
v
3 must reside within the first three
domains of CYR61.

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[in a new window]
|
Fig. 9.
Integrin
v 3
mediates CYR61 and CYR61 CT potentiation of
bFGF-induced DNA synthesis in 1064SK fibroblasts. Cells were
plated on 24-well dishes, serum-starved, and treated for 1 h at
room temperature with the indicated reagents before being stimulated
with bFGF (10 ng/ml), CYR61 (3 µg/ml), or CYR61 CT (3 µg/ml) and
[3H]thymidine in 0.5% BSA-containing serum-free medium
for 21 h. Incorporation assays were subsequently conducted as
described under "Materials and Methods." A, fibroblasts
were treated with GRGDSP peptide (0.2 mM), GRGESP peptide
(0.2 mM), or echistatin (1 µM). B,
10 µ g/ml anti- v 5 mAb clone P1F6,
anti- v mAb clone AV1, or anti- 3 mAb clone
B3A was used. C, cells were treated with
anti- v 3 mAb clone LM609 or a control
mouse IgG (10 µg/ml each). Data shown for all panels are mean ± S.D. of triplicate determinations and are representative of three
experiments.
|
|
 |
DISCUSSION |
The principal findings that emerged from this study provide new
insights into the structure-function relationship and mechanisms of
actions of the angiogenic inducer CYR61. Structurally, the CT domain of
CYR61 is dispensable for its chemotactic and mitogenesis enhancing
activities upon fibroblasts but is essential for its ability to support
fibroblast adhesion. Mechanistically, CYR61 induces fibroblast
migration through integrin
v
5 and
enhances growth factor-induced mitogenesis through integrin
v
3. Furthermore, we establish that CYR61
is a novel ligand for integrin
v
5. These findings demonstrate that CYR61 selectively utilizes distinct integrin
receptors to mediate disparate cellular activities and help to dissect
the functional roles of the structural domains in members of the CCN
protein family.
We have characterized the activities of CYR61
CT, a mutant that
deletes precisely the CT domain, compared with those of wild type
CYR61. Several reasons prompted us to focus on a deletion of the CT
domain in this study. First, Cop1/WISP2, a member of the CCN protein
family, lacks precisely the CT domain but is otherwise equally
homologous to other members of the family in the remaining three
domains (4, 5). This suggested to us that a CCN protein with only the
first three domains intact is likely capable of a subset of the
full-length protein functions. Second, a proteolytically processed
portion of CTGF corresponding to the CT domain alone can be isolated
from porcine uterine flushings (18). This CT domain fragment is
mitogenic and supports cell adhesion (32), providing evidence that CCN
family members may undergo limited proteolysis into biologically active
fragments in vivo (33, 34).
Inasmuch as the heparin binding activity contained within the CYR61 CT
domain is crucial for fibroblast adhesion (17), it is not surprising
that CYR61
CT is incapable of supporting fibroblast adhesion (Fig.
3). However, it is somewhat unexpected that CYR61
CT is nearly
identical to wild type CYR61 in its ability to stimulate fibroblast
migration and to enhance growth factor-induced mitogenesis (Fig. 4, 8),
thus demonstrating that the CT domain is non-essential for these
activities. Since the CT domain of CTGF has been reported to be
mitogenic (18), our results raise the possibility that mitogenesis
enhancing activities may reside in both the CT domain as well as within
the first three domains. However, whereas CTGF has been found to be
mitogenic by itself (35-39), CYR61 is not mitogenic on its own under
serum-free conditions but instead enhances the proliferative response
induced by other growth factors (6). Likewise, it has also been shown
that CTGF can act like CYR61 under similar assay conditions to augment
growth factor-induced mitogenesis (7). Thus, it is possible that the
reported mitogenic activities of CTGF and the mitogenesis enhancing
activity of CYR61 and CTGF are fundamentally distinct and may proceed
through different mechanisms. In this regard, we have shown that CYR61
potentiates growth factor-induced mitogenesis through integrin
v
3 (Fig. 9). The mechanism by which CTGF
acts mitogenically remains yet to be characterized.
The finding that the cell migration and mitogenesis enhancement
activities of CYR61 are dependent on the integrins
v
5 and
v
3,
respectively, is consistent with the known activities of these integrin
receptors to mediate cell migration and mitogenesis (25, 27, 40, 41).
In particular, signaling mediated through integrin
v
3 has been shown to activate components
of proliferative responses and converge with signaling pathways
initiated by mitogenic growth factors (42-44). Thus, the observation
that CYR61 enhances growth factor-induced mitogenesis, rather than
activating the mitogenic pathway on its own, is in keeping with its
mechanism of action through integrin
v
3.
Whereas CYR61 has been shown to be a direct ligand of integrins
v
3 and
IIb
3
(15, 16), we establish in this study that CYR61 is also a ligand for
integrin
v
5 (Fig. 7), which mediates the
chemotactic activity of CYR61 in fibroblasts (Fig. 6). Each of these
integrins recognizes protein ligands that contain an RGD recognition
sequence, and their interaction with ligands can be inhibited by
RGD-containing peptides, although non-RGD containing ligands are also
known (45-47). Neither CYR61 nor other CCN proteins possess a
canonical RGD-binding site (1), and the binding sites in CYR61 for
these integrins are currently unknown. Since CYR61
CT is able to
interact with integrins
v
3 and
v
5, binding sites for these integrins
must exist within the first three domains.
Originally identified as an immediate-early gene, the
CYR61 gene is rapidly induced by serum, bFGF,
platelet-derived growth factor, and transforming growth factor-
without requiring de novo protein synthesis (1, 48). CYR61
synthesis is highly induced in fibroblasts of granulation tissue during
wound healing (12, 49), possibly as a result of induction by growth
factors and cytokines that are up-regulated during wound repair
(50-52). The finding that the activities of CYR61 can be attributed to its interaction with integrin receptors is consistent with the up-regulation of several relevant integrins, including
v
5 and
v
3,
in granulation tissue during cutaneous wound healing (53-55). In this
context, CYR61 may synergize with the effects of growth factors by
acting as an angiogenic factor upon endothelial cells and as a
chemotactic and mitogenesis-enhancing factor upon fibroblasts. CYR61
can also induce the synthesis of matrix metalloproteinases 1 and 3 (10), enzymes known to play a role in angiogenesis and matrix
remodeling during wound repair (56-59). Since both angiogenesis and
wound healing occur in a protease-rich milieu, it is likely that
extracellular proteins such as CYR61 may be proteolytically processed.
The detection of biologically active fragments of CTGF provide evidence
for such processing events (18). The possibility that CYR61 and other
CCN proteins might be designed to maintain certain biological function
after proteolysis is intriguing. A cleavage product corresponding to
CYR61
CT, which is capable of stimulating fibroblast migration and
enhancing mitogenesis, will likely differ from full-length CYR61 by
being more diffusable as a results of having lost its heparin-binding
domain. Inasmuch as proteolytically processed fragments of CYR61 have
yet to be detected in vivo, these possibilities are
currently hypothetical and merit further investigation.
A remarkable finding of this study is that CYR61 utilizes distinct
integrin receptors for mediating different activities. We have
previously determined that fibroblast adhesion to CYR61 is mediated
through both integrin
6
1 and cell surface
heparan sulfate proteoglycans, which act as co-receptors (17).
Interfering with either receptor abolishes fibroblast adhesion to
CYR61. Surprisingly, neither receptor system appears to play any role
in mediating the chemotactic or mitogenesis enhancing activities of
CYR61. Instead, these activities are mediated through integrins
v
5 and
v
3,
respectively. Although many integrin ligands can bind more than one
integrin receptor, there are only a few instances where differential
integrin engagement in a specific cell type have been documented to
achieve functionally distinct outcomes. Examples include bone
sialoprotein, which supports human breast cancer cell adhesion and
proliferation through integrin
v
5 and migration through
v
3 (60). Similarly,
astrocyte adhesion to vitronectin is mediated through integrin
v
5, whereas migration proceeds through
integrin
v
8 (61). Notably, these examples all involve
v integrins. CYR61 is unique in being able
to interact with integrins
6
1,
v
5, and
v
3
in a functionally non-overlapping manner in human dermal fibroblasts.
The differential utilization of distinct integrin receptor by CYR61 may
be context-specific, as suggested by the experimental systems employed
to assay the activities involved. For example, whereas cell adhesion
was measured when CYR61 was immobilized onto plastic surfaces, the
chemotactic and mitogenesis enhancing activities were measured when
CYR61 was added as a soluble factor. Thus, immobilized CYR61 may
support cell adhesion when tethered to the ECM, whereas diffusable
CYR61, generated when dissociated from the ECM, may act as a
chemotactic or mitogenesis-enhancing factor through distinct integrin
receptors. In addition, integrin utilization by CYR61 appears to be
cell type-specific as well. For example, in contrast to fibroblasts,
cell adhesion and migration to CYR61 in endothelial cells are mediated
through integrin
v
3 (9, 15), and adhesion
of activated blood platelets is mediated through integrin
IIb
3 (16). The context- and cell
type-specific engagement of distinct integrin receptors to mediate
different activities by CYR61 provides an attractive mechanistic
paradigm for understanding how CCN proteins may act. The functional
diversity of CYR61 and other CCN proteins may thus reflect the
combinatorial activation of multiple integrin signaling systems in
different cell types and biological contexts.
 |
ACKNOWLEDGEMENTS |
We thank Stephen Lam and Chih-Chiun Chen for
helpful discussions and critical reading of the manuscript. The Munin
Corporation is the recipient of an SBIR grant.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grants CA46565, CA80080 (to L. F. L.), and CA78044.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 Molecular
Genetics, University of Illinois College of Medicine, 900 S. Ashland
Ave., Chicago, IL 60607. Tel.: 312-996-6978; Fax: 312-996-7034; E-mail:
lflau@uic.edu.
Published, JBC Papers in Press, April 3, 2001, DOI 10.1074/jbc.M100978200
 |
ABBREVIATIONS |
The abbreviations used are:
ECM, extracellular
matrix;
bFGF, basic fibroblast growth factor;
BSA, bovine serum
albumin;
CTGF, connective tissue growth factor;
mAb, monoclonal
antibody;
ELISA, enzyme-linked immunosorbent assay;
PMSF, phenylmethylsulfonyl fluoride;
CT, carboxyl-terminal.
 |
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