CYR61 Stimulates Human Skin Fibroblast Migration through Integrin alpha vbeta 5 and Enhances Mitogenesis through Integrin alpha vbeta 3, Independent of Its Carboxyl-terminal Domain*

Tatiana M. GrzeszkiewiczDagger , Deborah J. Kirschling§, Ningyu ChenDagger , and Lester F. LauDagger

From the Dagger  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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha 6beta 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 (CYR61Delta 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 alpha vbeta 5 but not integrins alpha 6beta 1 or alpha vbeta 3. Furthermore, we show that CYR61 binds directly to purified integrin alpha vbeta 5 in vitro. By contrast, CYR61 enhancement of basic fibroblast growth factor-induced DNA synthesis is mediated through integrin alpha vbeta 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 alpha 6beta 1, alpha vbeta 5, and alpha vbeta 3, respectively. Together, these findings establish CYR61 as a novel ligand for integrin alpha vbeta 5 and show that CYR61 interacts with distinct integrins to mediate disparate activities in a cell type-specific manner.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha vbeta 3 and alpha IIbbeta 3. Interaction between these integrins and CYR61 mediates endothelial cell adhesion and migration (integrin alpha vbeta 3) or blood platelet adhesion (integrin alpha IIbbeta 3) (9, 15, 16). CYR61 also supports the adhesion of primary human fibroblasts through integrin alpha 6beta 1 and cell surface heparan sulfate proteoglycans (17), resulting in adhesive signaling manifested by persistent formation of filopodia and lamellipodia, formation of integrin subunits alpha 6- and beta 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 (CYR61Delta 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 alpha 6beta 1 and heparan sulfate proteoglycans, CYR61-dependent chemotaxis and enhancement of DNA synthesis are mediated through integrins alpha vbeta 5 and alpha vbeta 3, respectively. We also show that CYR61 binds directly to purified integrin alpha vbeta 5 in vitro. These findings establish CYR61 as a novel ligand for integrin alpha vbeta 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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Antibodies, Peptides, and Reagents-- Function-blocking mAbs against integrins were purchased from Chemicon, Inc., including NKI-GoH3 (anti-alpha 6), NKI-M9 and AV1 (anti-alpha V), LM609 (anti-alpha Vbeta 3), P1F6 (anti-alpha Vbeta 5), JB1A (anti-beta 1), and B3A (anti-beta 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 CYR61Delta CT-- A construct encoding a CYR61 deletion mutant removing the CT domain, CYR61Delta 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 CYR61Delta CT construct was confirmed by DNA sequencing.

A recombinant baculovirus stock was generated as described previously (6). Recombinant CYR61Delta 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 CYR61Delta 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 CYR61Delta 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 alpha Vbeta 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 alpha Vbeta 5 was detected via ELISA adapted from a study of CYR61 binding to integrin alpha Vbeta 3 (15). Microtiter wells were coated with 50 µl/well of integrin alpha Vbeta 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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 (CYR61Delta 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, CYR61Delta 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 CYR61Delta CT proteins were produced in the baculovirus system and purified using the histidine tag via nickel-agarose column chromatography (22). The histidine-tagged CYR61Delta 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.   CYR61Delta CT protein purification. A, 30 µl of nickel-agarose-purified CYR61Delta 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 CYR61Delta 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.

To analyze the functional capabilities of CYR61Delta 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). CYR61Delta 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 CYR61Delta CT to coat the plastic microtiter wells. When wells were coated with either wild type CYR61 or CYR61Delta 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.   CYR61Delta 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.

Integrin alpha vbeta 5 Mediates Human Skin Fibroblast Migration to CYR61 and CYR61Delta 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 alpha vbeta 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 CYR61Delta 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 CYR61Delta 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 CYR61Delta 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 alpha 6beta 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-alpha 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 CYR61Delta CT (2 µg/ml). B, cells were treated with anti-beta 1 mAb clone JB1A (50 µg/ml) for 1 h prior to chamber loading and exposure to BSA (0.5%), CYR61 (2 µg/ml), CYR61Delta 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.

Since fibroblast adhesion to CYR61 is mediated through integrin alpha 6beta 1 and cell surface heparan sulfate proteoglycans function as co-receptors (17), we investigated whether integrin alpha 6beta 1 was responsible for CYR61-stimulated fibroblast migration. Cells were preincubated with either a mAb against integrin alpha 6 (GoH3) or integrin beta 1 (JB1A) for 1 h prior to cell migration assay. Neither mAb had any effect on cell migration to either CYR61 or CYR61Delta 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 alpha 5beta 1), but not vitronectin (which binds integrins alpha vbeta 3 and alpha vbeta 5), was completely blocked by mAb JB1A directed against the integrin beta 1 subunit. These results indicate that unlike fibroblast adhesion, fibroblast migration to CYR61 is not mediated through integrin alpha 6beta 1.

To elucidate the mechanism responsible for CYR61-stimulated fibroblast migration, we next focused on the alpha v integrins since CYR61 has been demonstrated to be a ligand of integrin alpha vbeta 3 (15). By using a mAb (NKI-M9) against the integrin alpha v subunit, we were able to inhibit CYR61- and CYR61Delta CT-stimulated migration (Fig. 6A), thus confirming the involvement of an alpha v integrin. As expected, NKI-M9 inhibited cell migration to vitronectin but not to fibronectin. LM609, a mAb with specificity for the integrin alpha vbeta 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 alpha vbeta 3. Since integrin alpha vbeta 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 alpha vbeta 5, migration to both CYR61 and CYR61Delta 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 alpha vbeta 5.


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Fig. 6.   Integrin alpha vbeta 5 mediates migration to CYR61 and CYR61Delta 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), CYR61Delta CT (2 µg/ml), fibronectin (FN) (10 ng/ml), or vitronectin (VN) (10 µg/ml). A, anti-alpha v mAb clone NKI-M9. B, anti-alpha vbeta 3 mAb clone LM609. C, anti-alpha vbeta 5 mAb clone P1F6. Data shown for all panels are mean ± S.D. of quadruplicate determinations and are representative of three experiments.

CYR61 Binds Directly to Integrin alpha vbeta 5 in Vitro-- We have shown previously that CYR61 is a ligand of, and binds directly to, integrins alpha vbeta 3 and alpha IIbbeta 3 (15, 16). That CYR61-stimulated fibroblast migration is mediated through integrin alpha vbeta 5 prompted us to investigate whether CYR61 can bind directly to this integrin. In a solid phase binding assay, purified integrin alpha vbeta 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 alpha vbeta 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 alpha vbeta 5. Microtiter wells were coated with purified alpha vbeta 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-alpha vbeta 5 mAb clone P1F6 (20 µg/ml), and anti-alpha vbeta 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.

To determine the specificity of the interaction between CYR61 and integrin alpha vbeta 5, inhibitors of integrin alpha vbeta 5 function were used as shown in Fig. 7B. The binding of CYR61 to immobilized alpha vbeta 5 was blocked by 5 mM EDTA and restored by the addition of 10 mM Mg2+, consistent with the divalent cation requirement of integrin alpha vbeta 5 (28, 29). Since integrin alpha vbeta 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 alpha vbeta 5. A function-blocking mAb against integrin alpha vbeta 5 (P1F6) inhibited CYR61 binding completely, whereas the anti-alpha vbeta 3 mAb (LM609) exhibited only a partial inhibitory effect. Taken together, these results show that CYR61 can bind directly to purified integrin alpha Vbeta 5 in vitro, consistent with functional studies showing that CYR61 stimulates fibroblast migration through this integrin (Fig. 7).

CYR61 and CYR61Delta CT Enhance bFGF-induced DNA Synthesis in Fibroblasts through Integrin alpha vbeta 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 CYR61Delta CT may have on 1064SK fibroblast proliferation. When added as a soluble factor to fibroblasts under serum-free conditions, CYR61Delta 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), CYR61Delta 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 CYR61Delta 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.


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Fig. 8.   CYR61Delta CT enhances bFGF-induced DNA synthesis in 1064SK fibroblasts. The effect of soluble CYR61Delta 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%), CYR61Delta 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 CYR61Delta 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 beta 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 alpha vbeta 3 (15), suggested to us that integrin alpha vbeta 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 alpha v (AV1) or beta 3 (B3A) completely abolished CYR61-enhanced mitogenesis but had no effect on bFGF-induced mitogenesis (Fig. 9B). However, a mAb against integrin alpha vbeta 5 (P1F6) did not have any effect, indicating that integrin alpha vbeta 5 does not play a role in CYR61 enhancement of mitogenesis. By contrast, LM609, a mAb directed against the integrin alpha vbeta 3 heterodimer, completely abolished CYR61- or CYR61Delta CT-enhanced mitogenesis, whereas the control IgG had no effect (Fig. 9C). Together, these results show that integrin alpha vbeta 3, but not integrin alpha vbeta 5, mediates the enhancement of bFGF-induced mitogenesis by CYR61. Moreover, CYR61Delta 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 alpha vbeta 3 must reside within the first three domains of CYR61.


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Fig. 9.   Integrin alpha vbeta 3 mediates CYR61 and CYR61Delta 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 CYR61Delta 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-alpha vbeta 5 mAb clone P1F6, anti-alpha v mAb clone AV1, or anti-beta 3 mAb clone B3A was used. C, cells were treated with anti-alpha vbeta 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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha vbeta 5 and enhances growth factor-induced mitogenesis through integrin alpha vbeta 3. Furthermore, we establish that CYR61 is a novel ligand for integrin alpha vbeta 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 CYR61Delta 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 CYR61Delta CT is incapable of supporting fibroblast adhesion (Fig. 3). However, it is somewhat unexpected that CYR61Delta 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 alpha vbeta 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 alpha vbeta 5 and alpha vbeta 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 alpha vbeta 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 alpha vbeta 3. Whereas CYR61 has been shown to be a direct ligand of integrins alpha vbeta 3 and alpha IIbbeta 3 (15, 16), we establish in this study that CYR61 is also a ligand for integrin alpha vbeta 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 CYR61Delta CT is able to interact with integrins alpha vbeta 3 and alpha vbeta 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-beta 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 alpha vbeta 5 and alpha vbeta 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 CYR61Delta 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 alpha 6beta 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 alpha vbeta 5 and alpha vbeta 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 alpha vbeta 5 and migration through alpha vbeta 3 (60). Similarly, astrocyte adhesion to vitronectin is mediated through integrin alpha vbeta 5, whereas migration proceeds through integrin alpha vbeta 8 (61). Notably, these examples all involve alpha v integrins. CYR61 is unique in being able to interact with integrins alpha 6beta 1, alpha vbeta 5, and alpha vbeta 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 alpha vbeta 3 (9, 15), and adhesion of activated blood platelets is mediated through integrin alpha IIbbeta 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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DISCUSSION
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