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• W2023397648 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 α6β1 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 (CYR61ΔCT) 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 αvβ5 but not integrins α6β1 or αvβ3. Furthermore, we show that CYR61 binds directly to purified integrin αvβ5 in vitro. By contrast, CYR61 enhancement of basic fibroblast growth factor-induced DNA synthesis is mediated through integrin αvβ3, 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 α6β1, αvβ5, and αvβ3, respectively. Together, these findings establish CYR61 as a novel ligand for integrin αvβ5 and show that CYR61 interacts with distinct integrins to mediate disparate activities in a cell type-specific manner. 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 α6β1 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 (CYR61ΔCT) 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 αvβ5 but not integrins α6β1 or αvβ3. Furthermore, we show that CYR61 binds directly to purified integrin αvβ5 in vitro. By contrast, CYR61 enhancement of basic fibroblast growth factor-induced DNA synthesis is mediated through integrin αvβ3, 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 α6β1, αvβ5, and αvβ3, respectively. Together, these findings establish CYR61 as a novel ligand for integrin αvβ5 and show that CYR61 interacts with distinct integrins to mediate disparate activities in a cell type-specific manner. extracellular matrix basic fibroblast growth factor bovine serum albumin connective tissue growth factor monoclonal antibody enzyme-linked immunosorbent assay phenylmethylsulfonyl fluoride carboxyl-terminal 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 (1Lau L.F. Lam S.C. Exp. Cell Res. 1999; 248: 44-57Crossref PubMed Scopus (578) Google Scholar, 2Brigstock D.R. Endocr. Rev. 1999; 20: 189-206Crossref PubMed Scopus (537) Google Scholar). 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) (3Bork P. FEBS Lett. 1993; 327: 125-130Crossref PubMed Scopus (549) Google Scholar). CCN family members contain all four conserved domains with the exception of WISP-2, which lacks precisely the carboxyl-terminal (CT) domain (4Zhang R. Averboukh L. Zhu W. Zhang H. Jo H. Dempsey P.J. Coffey R.J. Pardee A.B. Liang P. Mol. Cell. Biol. 1998; 18: 6131-6141Crossref PubMed Scopus (131) Google Scholar, 5Pennica D. Swanson T.A. Welsh J.W. Roy M.A. Lawrence D.A. Lee J. Brush J. Taneyhill L.A. Deuel B. Lew M. Watanabe C. Cohen R.L. Melhem M.F. Finley G.G. Quirke P. Goddard A.D. Hillan K.J. Gurney A.L. Botstein D. Levine A.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14717-14722Crossref PubMed Scopus (456) Google Scholar). 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 (6Kireeva M.L. Mo F.-E. Yang G.P. Lau L.F. Mol. Cell. Biol. 1996; 16: 1326-1334Crossref PubMed Scopus (303) Google Scholar, 7Kireeva M.L. Latinkic B.V. Kolesnikova T.V. Chen C.-C. Yang G.P. Abler A.S. Lau L.F. Exp. Cell Res. 1997; 233: 63-77Crossref PubMed Scopus (230) Google Scholar). In micromass cultures of mouse limb bud mesenchymal cells, CYR61 accelerates and promotes chondrogenic differentiation (8Wong M. Kireeva M.L. Kolesnikova T.V. Lau L.F. Dev. Biol. 1997; 192: 492-508Crossref PubMed Scopus (132) Google Scholar). CYR61 induces angiogenesis in vivo and enhances the tumorigenicity of human tumor cells in immunodeficient mice by increasing tumor size and vascularization (9Babic A.M. Kireeva M.L. Kolesnikova T.V. Lau L.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6355-6360Crossref PubMed Scopus (427) Google Scholar). CYR61 also up-regulates expression of matrix metalloproteinases 1 and 3 (10Chen C.-C. Chen N. Lau L.F. J. Biol. Chem. 2000; 276: 10443-10452Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar), 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 (11O'Brien T.P. Lau L.F. Cell Growth Differ. 1992; 3: 645-654PubMed Google Scholar, 12Latinkic, B. V., Mo, F.-E., Greenspan, J. A., Copeland, N. G., Gilbert, D. J., Jenkins, N. A., and Lau, L. F. (2001) Endocrinology, in press.Google Scholar) and in granulation tissue during cutaneous wound healing (7Kireeva M.L. Latinkic B.V. Kolesnikova T.V. Chen C.-C. Yang G.P. Abler A.S. Lau L.F. Exp. Cell Res. 1997; 233: 63-77Crossref PubMed Scopus (230) Google Scholar).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 (13Schwartz M.A. Schaller M.D. Ginsberg M.H. Annu. Rev. Cell Dev. Biol. 1995; 11: 549-599Crossref PubMed Scopus (1461) Google Scholar,14Hynes R.O. Dev. Biol. 1996; 180: 402-412Crossref PubMed Scopus (250) Google Scholar). Recombinant CYR61 protein is a ligand of, and binds directly to, integrins αvβ3 and αIIbβ3. Interaction between these integrins and CYR61 mediates endothelial cell adhesion and migration (integrin αvβ3) or blood platelet adhesion (integrin αIIbβ3) (9Babic A.M. Kireeva M.L. Kolesnikova T.V. Lau L.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6355-6360Crossref PubMed Scopus (427) Google Scholar, 15Kireeva M.L. Lam S.C.T. Lau L.F. J. Biol. Chem. 1998; 273: 3090-3096Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 16Jedsadayanmata A. Chen C.C. Kireeva M.L. Lau L.F. Lam S.C. J. Biol. Chem. 1999; 274: 24321-24327Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). CYR61 also supports the adhesion of primary human fibroblasts through integrin α6β1 and cell surface heparan sulfate proteoglycans (17Chen N. Chen C.C. Lau L.F. J. Biol. Chem. 2000; 275: 24953-24961Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar), resulting in adhesive signaling manifested by persistent formation of filopodia and lamellipodia, formation of integrin subunits α6- and β1-containing focal complexes, activation of focal adhesion kinase and mitogen-activated protein kinases, and up-regulation of matrix metalloproteinases 1 and 3 (10Chen C.-C. Chen N. Lau L.F. J. Biol. Chem. 2000; 276: 10443-10452Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar).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 (18Brigstock D.R. Steffen C.L. Kim G.Y. Vegunta R.K. Diehl J.R. Harding P.A. J. Biol. Chem. 1997; 272: 20275-20282Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). To investigate the structure-function relationship of CCN proteins, we have constructed a mutant form of CYR61 (CYR61ΔCT) 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 α6β1 and heparan sulfate proteoglycans, CYR61-dependent chemotaxis and enhancement of DNA synthesis are mediated through integrins αvβ5 and αvβ3, respectively. We also show that CYR61 binds directly to purified integrin αvβ5 in vitro. These findings establish CYR61 as a novel ligand for integrin αvβ5, identify a functional division within the modular structure of the protein, and demonstrate that different integrin receptors mediate distinct activities of CYR61.RESULTSThe 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 (CYR61ΔCT) 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, CYR61ΔCT 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 CYR61ΔCT proteins were produced in the baculovirus system and purified using the histidine tag via nickel-agarose column chromatography (22Dhanabal M. Fryxell D.K. Ramakrishnan S. J. Immunol. Methods. 1995; 182: 165-175Crossref PubMed Scopus (11) Google Scholar). The histidine-tagged CYR61ΔCT bound well to the nickel column, allowing its purification to near homogeneity as judged by SDS-polyacrylamide gel electrophoresis (Fig. 2 A) and immunoblotting with affinity-purified anti-CYR61 antibodies (Fig. 2 B).Figure 2CYR61Δ CT protein purification. A, 30 μl of nickel-agarose-purified CYR61ΔCT 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 CYR61ΔCT 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To analyze the functional capabilities of CYR61ΔCT, 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. 3 A), consistent with previous studies (17Chen N. Chen C.C. Lau L.F. J. Biol. Chem. 2000; 275: 24953-24961Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). CYR61ΔCT 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 CYR61ΔCT to coat the plastic microtiter wells. When wells were coated with either wild type CYR61 or CYR61ΔCT, both bound proteins could be readily detected to similar levels with anti-CYR61 polyclonal antibodies by ELISA (Fig.3 B). 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 (17Chen N. Chen C.C. Lau L.F. J. Biol. Chem. 2000; 275: 24953-24961Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar).Integrin αvβ5 Mediates Human Skin Fibroblast Migration to CYR61 and CYR61ΔCTWe have previously found that CYR61 promotes migration of both fibroblasts and endothelial cells and that endothelial cell chemotaxis to CYR61 is mediated through integrin αvβ3 (6Kireeva M.L. Mo F.-E. Yang G.P. Lau L.F. Mol. Cell. Biol. 1996; 16: 1326-1334Crossref PubMed Scopus (303) Google Scholar, 9Babic A.M. Kireeva M.L. Kolesnikova T.V. Lau L.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6355-6360Crossref PubMed Scopus (427) Google Scholar). By using a modified Boyden chamber assay, we investigated whether the CT domain was required for fibroblast migration. As shown in Fig.4 A, 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 CYR61ΔCT 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 (17Chen N. Chen C.C. Lau L.F. J. Biol. Chem. 2000; 275: 24953-24961Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). Since the CYR61ΔCT 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.5 A). Therefore, the CT domain of CYR61 and its heparin-binding capability are not required for CYR61-stimulated fibroblast migration.Figure 4The 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 CYR61ΔCT. C, migration to CYR61DM. Data shown for all panels are mean ± S.D. of quadruplicate determinations and are representative of three experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 5Migration to CYR61 protein is not mediated by integrin α6β1or 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-α6mAb 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 CYR61ΔCT (2 μg/ml). B, cells were treated with anti-β1 mAb clone JB1A (50 μg/ml) for 1 h prior to chamber loading and exposure to BSA (0.5%), CYR61 (2 μg/ml), CYR61ΔCT (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.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Since fibroblast adhesion to CYR61 is mediated through integrin α6β1 and cell surface heparan sulfate proteoglycans function as co-receptors (17Chen N. Chen C.C. Lau L.F. J. Biol. Chem. 2000; 275: 24953-24961Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar), we investigated whether integrin α6β1 was responsible for CYR61-stimulated fibroblast migration. Cells were preincubated with either a mAb against integrin α6 (GoH3) or integrin β1 (JB1A) for 1 h prior to cell migration assay. Neither mAb had any effect on cell migration to either CYR61 or CYR61ΔCT (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 α5β1), but not vitronectin (which binds integrins αvβ3 and αvβ5), was completely blocked by mAb JB1A directed against the integrin β1 subunit. These results indicate that unlike fibroblast adhesion, fibroblast migration to CYR61 is not mediated through integrin α6β1.To elucidate the mechanism responsible for CYR61-stimulated fibroblast migration, we next focused on the αv integrins since CYR61 has been demonstrated to be a ligand of integrin αvβ3 (15Kireeva M.L. Lam S.C.T. Lau L.F. J. Biol. Chem. 1998; 273: 3090-3096Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). By using a mAb (NKI-M9) against the integrin αv subunit, we were able to inhibit CYR61- and CYR61ΔCT-stimulated migration (Fig.6 A), thus confirming the involvement of an αv integrin. As expected, NKI-M9 inhibited cell migration to vitronectin but not to fibronectin. LM609, a mAb with specificity for the integrin αvβ3 heterodimer (23Cheresh D.A. Spiro R.C. J. Biol. Chem. 1987; 262: 17703-17711Abstract Full Text PDF PubMed Google Scholar), had no effect on cell migration to CYR61 but blocked migration to vitronectin as expected (Fig. 6 B). Therefore, in contrast to migration in endothelial cells (9Babic A.M. Kireeva M.L. Kolesnikova T.V. Lau L.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6355-6360Crossref PubMed Scopus (427) Google Scholar), CYR61-stimulated fibroblast migration is not mediated through integrin αvβ3. Since integrin αvβ5 is expressed in fibroblasts and has been shown to mediate activation-dependent cell migration (24Cavani A. Zambruno G. Marconi A. Manca V. Marchetti M. Giannetti A. J. Invest Dermatol. 1993; 101: 600-604Abstract Full Text PDF PubMed Google Scholar, 25Kim J.P. Zhang K. Chen J.D. Kramer R.H. Woodley D.T. J. Biol. Chem. 1994; 269: 26926-26932Abstract Full Text PDF PubMed Google Scholar, 26Gailit J. Welch M.P. Clark R.A. J. Invest Dermatol. 1994; 103: 221-227Abstract Full Text PDF PubMed Scopus (196) Google Scholar, 27Klemke R.L. Yebra M. Bayna E.M. Cheresh D.A. J. Cell Biol. 1994; 127: 859-866Crossref PubMed Scopus (248) Google Scholar), we examined its ability to mediate CYR61-induced migration. When fibroblasts were treated with P1F6, a mAb against integrin αvβ5, migration to both CYR61 and CYR61ΔCT protein was blocked completely (Fig. 6 C). As expected, P1F6 had no effect on migration to fibronectin. Together, these results show that fibroblast migration to CYR61 is mediated through integrin αvβ5.Figure 6Integrin αvβ5mediates migration to CYR61 and CYR61Δ 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), CYR61ΔCT (2 μg/ml), fibronectin (FN) (10 ng/ml), or vitronectin (VN) (10 μg/ml). A, anti-αv mAb clone NKI-M9. B, anti-αvβ3 mAb clone LM609. C, anti-αvβ5 mAb clone P1F6. Data shown for all panels are mean ± S.D. of quadruplicate determinations and are representative of three experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)CYR61 Binds Directly to Integrin αvβ5in VitroWe have shown previously that CYR61 is a ligand of, and binds directly to, integrins αvβ3 and αIIbβ3 (15Kireeva M.L. Lam S.C.T. Lau L.F. J. Biol. Chem. 1998; 273: 3090-3096Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 16Jedsadayanmata A. Chen C.C. Kireeva M.L. Lau L.F. Lam S.C. J. Biol. Chem. 1999; 274: 24321-24327Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). That CYR61-stimulated fibroblast migration is mediated through integrin αvβ5 prompted us to investigate whether CYR61 can bind directly to this integrin. In a solid phase binding assay, purified integrin αvβ5 receptor was immobilized on microtiter wells onto which CYR61 was allowed to bind. The reaction was detected by ELISA using anti-CYR61 antibodies. Fig.7 A shows that CYR61 bound to immobilized integrin αvβ5 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.Figure 7CYR61 binds directly to immobilized integrin αvβ5.Microtiter wells were coated with purified αvβ5 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-αvβ5 mAb clone P1F6 (20 μg/ml), and anti-αvβ3 mAb clone LM609 (20 μg/ml). Data shown for all panels are mean ± S.D. of triplicate determinations and are representative of three experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To determine the specificity of the interaction between CYR61 and integrin αvβ5, inhibitors of integrin αvβ5 function were used as shown in Fig.7 B. The binding of CYR61 to immobilized αvβ5 was blocked by 5 mm EDTA and restored by the addition of 10 mm Mg2+, consistent with the divalent cation requirement of integrin αvβ5 (28Stuiver I. Ruggeri Z. Smith J.W. J. Cell. Physiol. 1996; 168: 521-531Crossref PubMed Scopus (23) Google Scholar, 29Hu D.D. Lin E.C. Kovach N.L. Hoyer J.R. Smith J.W. J. Biol. Chem. 1995; 270: 26232-26238Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar). Since integrin αvβ5 recognizes protein ligands with the RGD sequence motif and is inhibited by soluble RGD peptides (30Plow E.F. Haas T.A. Zhang L. Loftus J. Smith J.W. J. Biol. Chem. 2000; 275: 21785-21788Abstract Full Text Full Text PDF PubMed Scopus (1100) Google Scholar), 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 αvβ5. A function-blocking mAb against integrin αvβ5(P1F6) inhibited CYR61 binding completely, whereas the anti-αvβ3 mAb (LM609) exhibited only a partial inhibitory effect. Taken together, these results show that CYR61 can bind directly to purified integrin αVβ5 in vitro, consistent with functional studies showing that CYR61 stimulates fibroblast migration through this integrin (Fig. 7).CYR61 and CYR61ΔCT Enhance bFGF-induced DNA Synthesis in Fibroblasts through Integrin αvβ3Although CYR61 is not mitogenic alone, its ability to enhance growth factor-induced DNA synthesis has been established for fibroblasts and endothelial cells (6Kireeva M.L. Mo F.-E. Yang G.P. Lau L.F. Mol. Cell. Biol. 1996; 16: 1326-1334Crossref PubMed Scopus (303) Google Scholar, 7Kireeva M.L. Latinkic B.V. Kolesnikova T.V. Chen C.-C. Yang G.P. Abler A.S. Lau L.F. Exp. Cell Res. 1997; 233: 63-77Crossref PubMed Scopus (230) Google Scholar). Thus, we were interested in elucidating what activity CYR61ΔCT may have on 1064SK fibroblast proliferation. When added as a soluble factor to fibroblasts under serum-free conditions, CYR61ΔCT alone did not elicit a mitogenic response (Fig.8 A) as described previously for wild type CYR61 (6Kireeva M.L. Mo F.-E. Yang G.P. Lau L.F. Mol. Cell. Biol. 1996; 16: 1326-1334Crossref PubMed Scopus (303) Google Scholar). However, when added in the presence of a suboptimal concentration of bFGF (10 ng/ml), CYR61ΔCT elicited a 2-fold increase over bFGF treatment in thymidine incorporation similar to wild type CYR61 (Fig. 8 A). Furthermore, the mitogenesis-enhancing effect of CYR61ΔCT is dose-dependent and saturable, with maximal potentiation occurring at 3 μg/ml under serum-free conditions (Fig.8 B). These studies show that the CT domain is not required for CYR61 to enhance growth factor-induced DNA synthesis.Figure 8CYR61Δ CT enhances bFGF-induced DNA synthesis in 1064SK fibroblasts. The effect of soluble CYR61ΔCT 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%), CYR61ΔCT (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 CYR61ΔCT, 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT)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.9 A). 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 β3 integrins (31Gan Z.R. Gould R.J. Jacobs J.W. Friedman P.A. Polokoff M.A. J. Biol. Chem. 1988; 263: 19827-19832Abstract Full Text PDF PubMed Google Scholar), CYR61 enhancement of bFGF-induced proliferation was similarly abrogated completely. These results, together with the fact that CYR61 is a known ligand of integrin αvβ3 (15Kireeva M.L. Lam S.C.T. Lau L.F. J. Biol. Chem. 1998; 273: 3090-3096Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar), suggested to us that integrin αvβ3 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 αv (AV1) or β3 (B3A) completely abolished CYR61-e" @default.
• W2023397648 created "2016-06-24" @default.
• W2023397648 creator A5011708451 @default.
• W2023397648 creator A5014230378 @default.
• W2023397648 creator A5024562124 @default.
• W2023397648 creator A5080362561 @default.
• W2023397648 date "2001-06-01" @default.
• W2023397648 modified "2023-10-14" @default.
• W2023397648 title "CYR61 Stimulates Human Skin Fibroblast Migration through Integrin αvβ5 and Enhances Mitogenesis through Integrin αvβ3, Independent of Its Carboxyl-terminal Domain" @default.
• W2023397648 cites W1521201537 @default.
• W2023397648 cites W1538887755 @default.
• W2023397648 cites W1541442773 @default.
• W2023397648 cites W1554896558 @default.
• W2023397648 cites W1577857859 @default.
• W2023397648 cites W1925481732 @default.
• W2023397648 cites W1955701221 @default.
• W2023397648 cites W1964941505 @default.
• W2023397648 cites W1970483666 @default.
• W2023397648 cites W1983871846 @default.
• W2023397648 cites W1989021197 @default.
• W2023397648 cites W1994724799 @default.
• W2023397648 cites W1996293220 @default.
• W2023397648 cites W1996534526 @default.
• W2023397648 cites W1999512677 @default.
• W2023397648 cites W2002740306 @default.
• W2023397648 cites W2005463323 @default.
• W2023397648 cites W2014326933 @default.
• W2023397648 cites W2032912914 @default.
• W2023397648 cites W2040283898 @default.
• W2023397648 cites W2041137384 @default.
• W2023397648 cites W2045243608 @default.
• W2023397648 cites W2047277690 @default.
• W2023397648 cites W2055970438 @default.
• W2023397648 cites W2057452914 @default.
• W2023397648 cites W2059427623 @default.
• W2023397648 cites W2059442542 @default.
• W2023397648 cites W2063242845 @default.
• W2023397648 cites W2072103796 @default.
• W2023397648 cites W2076628456 @default.
• W2023397648 cites W2080144763 @default.
• W2023397648 cites W2085804760 @default.
• W2023397648 cites W2087342203 @default.
• W2023397648 cites W2088993413 @default.
• W2023397648 cites W2090619182 @default.
• W2023397648 cites W2098964232 @default.
• W2023397648 cites W2104373491 @default.
• W2023397648 cites W2104762951 @default.
• W2023397648 cites W2112417973 @default.
• W2023397648 cites W2133341222 @default.
• W2023397648 cites W2133433848 @default.
• W2023397648 cites W2158545941 @default.
• W2023397648 cites W2162982642 @default.
• W2023397648 cites W2167807541 @default.
• W2023397648 cites W2168918180 @default.
• W2023397648 cites W2169804478 @default.
• W2023397648 cites W2325727928 @default.
• W2023397648 cites W2410336415 @default.
• W2023397648 cites W4230129623 @default.
• W2023397648 cites W4232957465 @default.
• W2023397648 cites W4239357337 @default.
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