The Angiogenic Factors Cyr61 and Connective Tissue Growth Factor Induce Adhesive Signaling in Primary Human Skin Fibroblasts

Abstract

The angiogenic inducers cysteine-rich angiogenic protein 61 (Cyr61) and connective tissue growth factor (CTGF) are structurally related, extracellular matrix-associated heparin-binding proteins. Both can stimulate chemotaxis and promote proliferation in endothelial cells and fibroblasts in culture and induce neovascularization in vivo. Encoded by inducible immediate early genes, Cyr61 and CTGF are synthesized upon growth factor stimulation in cultured fibroblasts and during cutaneous wound healing in dermal fibroblasts. Recently, we have shown that adhesion of primary human fibroblasts to immobilized Cyr61 is mediated through integrin α6β1 and cell surface heparan sulfate proteoglycans (HSPGs) (Chen, N., Chen, C.-C., and Lau, L.F. (2000) J. Biol. Chem. 275, 24953–24961), providing the first demonstration of an absolute requirement for HSPGs in integrin-mediated cell attachment. We show in this study that CTGF also mediates fibroblast adhesion through the same mechanism and demonstrate that fibroblasts adhesion to immobilized Cyr61 or CTGF induces distinct adhesive signaling responses consistent with their biological activities. Compared with fibroblast adhesion to fibronectin, laminin, or type I collagen, cell adhesion to Cyr61 or CTGF induces 1) more extensive and prolonged formation of filopodia and lamellipodia, concomitant with formation of integrin α6β1-containing focal complexes localized at leading edges of pseudopods; 2) activation of intracellular signaling molecules including focal adhesion kinase, paxillin, and Rac with similar rapid kinetics; 3) sustained activation of p42/p44 MAPKs lasting for at least 9 h; and 4) prolonged gene expression changes including up-regulation of MMP-1 (collagenase-1) and MMP-3 (stromelysin-1) mRNAs and proteins sustained for at least 24 h. Together, these results establish Cyr61 and CTGF as bona fide adhesive substrates with specific signaling capabilities, provide a molecular basis for their activities in fibroblasts through integrin α6β1 and HSPG-mediated signaling during attachment and indicate that these proteins may function in matrix remodeling through the activation of metalloproteinases during angiogenesis and wound healing. The angiogenic inducers cysteine-rich angiogenic protein 61 (Cyr61) and connective tissue growth factor (CTGF) are structurally related, extracellular matrix-associated heparin-binding proteins. Both can stimulate chemotaxis and promote proliferation in endothelial cells and fibroblasts in culture and induce neovascularization in vivo. Encoded by inducible immediate early genes, Cyr61 and CTGF are synthesized upon growth factor stimulation in cultured fibroblasts and during cutaneous wound healing in dermal fibroblasts. Recently, we have shown that adhesion of primary human fibroblasts to immobilized Cyr61 is mediated through integrin α6β1 and cell surface heparan sulfate proteoglycans (HSPGs) (Chen, N., Chen, C.-C., and Lau, L.F. (2000) J. Biol. Chem. 275, 24953–24961), providing the first demonstration of an absolute requirement for HSPGs in integrin-mediated cell attachment. We show in this study that CTGF also mediates fibroblast adhesion through the same mechanism and demonstrate that fibroblasts adhesion to immobilized Cyr61 or CTGF induces distinct adhesive signaling responses consistent with their biological activities. Compared with fibroblast adhesion to fibronectin, laminin, or type I collagen, cell adhesion to Cyr61 or CTGF induces 1) more extensive and prolonged formation of filopodia and lamellipodia, concomitant with formation of integrin α6β1-containing focal complexes localized at leading edges of pseudopods; 2) activation of intracellular signaling molecules including focal adhesion kinase, paxillin, and Rac with similar rapid kinetics; 3) sustained activation of p42/p44 MAPKs lasting for at least 9 h; and 4) prolonged gene expression changes including up-regulation of MMP-1 (collagenase-1) and MMP-3 (stromelysin-1) mRNAs and proteins sustained for at least 24 h. Together, these results establish Cyr61 and CTGF as bona fide adhesive substrates with specific signaling capabilities, provide a molecular basis for their activities in fibroblasts through integrin α6β1 and HSPG-mediated signaling during attachment and indicate that these proteins may function in matrix remodeling through the activation of metalloproteinases during angiogenesis and wound healing. connective tissue growth factor basic fibroblast growth factor bovine serum albumin extracellular matrix focal adhesion kinase heparan sulfate proteoglycan Iscove's modified Dulbecco's medium mitogen-activated protein kinase matrix metalloproteinase monoclonal antibodies phosphate buffered saline transforming growth factor-β1 Wnt-inducible secreted protein polyacrylamide gel electrophoresis. The recent emergence of the CCN family of angiogenic regulators has called attention to their functional versatility and mechanisms of actions (1Lau L.F. Lam S.C. Exp. Cell Res. 1999; 248: 44-57Crossref PubMed Scopus (580) Google Scholar). This family of secreted proteins consists of six members:Cyr61,CTGF1,Nov, WISP-1, WISP-2, and WISP-3 (1Lau L.F. Lam S.C. Exp. Cell Res. 1999; 248: 44-57Crossref PubMed Scopus (580) Google Scholar, 2Brigstock D.R. Endocr. Rev. 1999; 20: 189-206Crossref PubMed Scopus (540) Google Scholar), with the first three members of the family identified providing the acronym CCN. These structurally conserved proteins share four modular domains with sequence similarities to insulin-like growth factor-binding proteins, von Willebrand factor type C repeat, thrombospondin type 1 repeat, and growth factor cysteine knots (1Lau L.F. Lam S.C. Exp. Cell Res. 1999; 248: 44-57Crossref PubMed Scopus (580) Google Scholar, 3Bork P. FEBS Lett. 1993; 327: 125-130Crossref PubMed Scopus (552) Google Scholar). Each of these domains is encoded by a separate exon, suggesting that CCN genes arose through exon shuffling of preexisting genes to form proteins with multiple functional domains. Cyr61 and CTGF are both encoded by immediate early genes and are coinduced by serum, bFGF, platelet-derived growth factor, and TGF-β1 in fibroblasts (4O'Brien T.P. Yang G.P. Sanders L. Lau L.F. Mol. Cell. Biol. 1990; 10: 3569-3577Crossref PubMed Scopus (271) Google Scholar, 5Lau L.F. Nathans D. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 1182-1186Crossref PubMed Scopus (639) Google Scholar, 6Ryseck R.-P. Macdonald-Bravo H. Mattei M.-G. Bravo R. Cell Growth Differ. 1991; 2: 225-233PubMed Google Scholar, 7Brunner A. Chinn J. Neubauer M. Purchio A.F. DNA Cell Biol. 1991; 10: 293-300Crossref PubMed Scopus (115) Google Scholar). Cyr61 and CTGF share ∼45% amino acid sequence identity (4O'Brien T.P. Yang G.P. Sanders L. Lau L.F. Mol. Cell. Biol. 1990; 10: 3569-3577Crossref PubMed Scopus (271) Google Scholar, 8Bradham D.M. Igarashi A. Potter R.L. Grotendorst G.R. J. Cell Biol. 1991; 114: 1285-1294Crossref PubMed Scopus (809) Google Scholar), and both proteins bind heparin, associate with the ECM, and exhibit remarkable functional versatility (9Yang G.P. Lau L.F. Cell Growth Differ. 1991; 2: 351-357PubMed Google Scholar, 10Kireeva 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 (231) Google Scholar). Purified Cyr61 and CTGF mediate cell adhesion, stimulate cell migration, and augment growth factor-induced DNA synthesis (10Kireeva 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 (231) Google Scholar, 11Kireeva M.L. Mo F.-E. Yang G.P. Lau L.F. Mol. Cell. Biol. 1996; 16: 1326-1334Crossref PubMed Scopus (304) Google Scholar, 12Frazier K. Williams S. Kothapalli D. Klapper H. Grotendorst G.R. J. Invest. Dermatol. 1996; 107: 404-411Abstract Full Text PDF PubMed Scopus (673) Google Scholar). Cyr61 and CTGF can promote chondrogenic differentiation, consistent with their expression in prechondrogenic mesenchyme during embryogenesis (13O'Brien T.P. Lau L.F. Cell Growth Differ. 1992; 3: 645-654PubMed Google Scholar, 14Wong M. Kireeva M.L. Kolesnikova T.V. Lau L.F. Dev. Biol. 1997; 192: 492-508Crossref PubMed Scopus (134) Google Scholar, 15Nakanishi T. Nishida T. Shimo T. Kobayashi K. Kubo T. Tamatani T. Tezuka K. Takigawa M. Endocrinology. 2000; 141: 264-273Crossref PubMed Scopus (214) Google Scholar). Both Cyr61 and CTGF stimulate chemotaxis in endothelial cells through an integrin αVβ3-dependent pathway and induce neovascularization in vivo (16Babic A.M. Kireeva M.L. Kolesnikova T.V. Lau L.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6355-6360Crossref PubMed Scopus (431) Google Scholar, 17Babic A.M. Chen C.-C. Lau L.F. Mol. Cell. Biol. 1999; 19: 2958-2966Crossref PubMed Google Scholar). Expression of Cyr61 in tumor cells enhances tumorigenicity by increasing tumor size and vascularization (16Babic A.M. Kireeva M.L. Kolesnikova T.V. Lau L.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6355-6360Crossref PubMed Scopus (431) Google Scholar), whereas expression of CTGF has been correlated with both systemic and localized fibrotic diseases (18Igarashi A. Nashiro K. Kikuchi K. Sato S. Ihn H. Grotendorst G.R. Takehara K. J. Invest. Dermatol. 1995; 105: 280-284Abstract Full Text PDF PubMed Scopus (261) Google Scholar, 19Igarashi A. Nashiro K. Kikuchi K. Sato S. Ihn H. Fujimoto M. Grotendorst G.R. Takehara K. J. Invest. Dermatol. 1996; 106: 729-733Abstract Full Text PDF PubMed Scopus (392) Google Scholar, 20Lasky J.A. Ortiz L.A. Tonthat B. Hoyle G.W. Corti M. Athas G. Lungarella G. Brody A. Friedman M. Am. J. Physiol. 1998; 275: L365-L371PubMed Google Scholar). Both proteins are also coinduced in granulation tissues during cutaneous wound healing (19Igarashi A. Nashiro K. Kikuchi K. Sato S. Ihn H. Fujimoto M. Grotendorst G.R. Takehara K. J. Invest. Dermatol. 1996; 106: 729-733Abstract Full Text PDF PubMed Scopus (392) Google Scholar, 21Latinkic B.V. Regulation of Expression of Growth Factor-inducible Immediate-Early Genes cyr61 and pip92 Ph.D. thesis. University of Illinois at Chicago, 1994Google Scholar). Thus, Cyr61 and CTGF may participate in wound repair by acting as angiogenic inducers upon endothelial cells and by acting as chemotactic, proliferative, and matrix remodeling factors upon fibroblasts. Through what mechanism(s) might Cyr61 and CTGF act to execute such a diversity of biological functions? Inasmuch as CTGF was first identified as a growth factor (8Bradham D.M. Igarashi A. Potter R.L. Grotendorst G.R. J. Cell Biol. 1991; 114: 1285-1294Crossref PubMed Scopus (809) Google Scholar), it has been tempting to postulate that it might function as a classical growth factor, although a cell surface receptor for CTGF that resembles a classical growth factor receptor has not been identified to date. By contrast, both Cyr61 and CTGF share characteristics of ECM-associated signaling proteins in several compelling ways (1Lau L.F. Lam S.C. Exp. Cell Res. 1999; 248: 44-57Crossref PubMed Scopus (580) Google Scholar, 22Bornstein P. J. Cell Biol. 1995; 130: 503-506Crossref PubMed Scopus (581) Google Scholar): 1) they are heparin-binding, ECM-associated proteins; 2) they contain sequence similarities to matrix proteins including von Willebrand factor and thrombospondin; and 3) they regulate cell adhesion, migration, proliferation, differentiation, and survival, all functions that can be modulated through cell and matrix interactions, especially through integrin receptors (23Schwartz M.A. Schaller M.D. Ginsberg M.H. Annu. Rev. Cell Dev. Biol. 1995; 11: 549-599Crossref PubMed Scopus (1467) Google Scholar, 24Hynes R.O. Dev. Biol. 1996; 180: 402-412Crossref PubMed Scopus (251) Google Scholar). Indeed, we have demonstrated that Cyr61 and CTGF are ligands of, and bind directly to, the integrins αVβ3 and αIIbβ3(25Kireeva M.L. Lam S.C.T. Lau L.F. J. Biol. Chem. 1998; 273: 3090-3096Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 26Jedsadayanmata 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 (193) Google Scholar). Recently, we showed that Cyr61 supports the attachment, or the early phase of cell adhesion, of human skin fibroblasts through integrin α6β1 and HSPGs (27Chen N. Chen C.C. Lau L.F. J. Biol. Chem. 2000; 275: 24953-24961Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). To understand their mechanism of actions, we have investigated the cellular responses to and signaling consequences of cell adhesion to immobilized Cyr61 and CTGF. We present herein the first conclusive evidence that Cyr61 and CTGF function as adhesive signaling molecules. In the absence of any other stimulus, fibroblast adhesion to either protein is sufficient to generate signaling events resulting in morphological changes, activation of intracellular kinases, and activation of gene expression consistent with the biological activities of these CCN proteins. These findings establish Cyr61 and CTGF as inducible, ECM-associated adhesive substrates capable of signaling through integrin-mediated pathways, provide a mechanistic interpretation for the chemotactic and mitogenic activities of these proteins, point to unique signaling capabilities of integrin α6β1 and HSPGs, and indicate a potential function for Cyr61 and CTGF in matrix remodeling through the activation of metalloproteinases during angiogenesis and wound healing. Normal human fibroblasts (1064SK) from skin biopsy of healthy newborn were obtained from the American Type Culture Collection (ATCC; number CRL-2076). The culture was maintained in IMDM (Life Technologies, Inc.) with 10% fetal bovine serum (Intergen, Purchase, NY) at 37 °C with 5% CO2 and used for experiments before passage 8. Cell adhesion assays were carried out largely as described (10Kireeva 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 (231) Google Scholar, 25Kireeva M.L. Lam S.C.T. Lau L.F. J. Biol. Chem. 1998; 273: 3090-3096Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). Briefly, 96-well microtiter plates (Becton Dickinson, Franklin Lakes, NJ) were coated with test proteins diluted in PBS (137 mm NaCl, 2.7 mmKCl, 4.3 mm Na2HPO4, 1.4 mm KH2PO4, pH 7.3) at 50 μl/well, incubated at 4 °C for 16 h, and blocked with 1% BSA at room temperature for 1 h. 1064SK fibroblasts were harvested in PBS with 2.5 mm EDTA and resuspended in IMDM with 0.5% BSA at 5 × 105 cells/ml and allowed to adhere to protein-coated wells at 50 μl/well. After incubation at 37 °C for 30 min, wells were washed twice with PBS. Adherent cells were fixed with 10% formalin, stained with methylene blue, and quantified by dye extraction and measurement of absorbance at 620 nm (28Oliver M.H. Harrison N.K. Bishop J.E. Cole P.J. Laurent G.J. J. Cell Sci. 1989; 92: 513-518Crossref PubMed Google Scholar). Where indicated, EDTA or peptides were mixed with cells prior to plating. Antibodies were incubated with cells at room temperature for 1 h before plating. Inhibition of glycosaminoglycan sulfation was achieved by growing cells in medium containing sodium chlorate (40 mm) for 24 h; cells were then detached and plated for cell adhesion assays as described above (29Rapraeger A.C. Krufka A. Olwin B.B. Science. 1991; 252: 1705-1708Crossref PubMed Scopus (1290) Google Scholar). To show the specificity of sulfation blockage, 10 mm sodium sulfate was included in the culture medium together with sodium chlorate. Murine Cyr61 and CTGF proteins were synthesized in a Baculovirus expression system (Invitrogen Corp., Carlsbad, CA) using Sf9 cells and purified from serum-free insect cell conditioned medium on Sepharose-S columns as described (10Kireeva 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 (231) Google Scholar, 11Kireeva M.L. Mo F.-E. Yang G.P. Lau L.F. Mol. Cell. Biol. 1996; 16: 1326-1334Crossref PubMed Scopus (304) Google Scholar). Fibronectin, laminin, vitronectin, and type I collagen were purchased from Collaborative Biomedical (Bedford, MA). BSA, heparinase I (EC 4.2.2.7), chondroitinase ABC (EC 4.2.2.4), and heparin (sodium salt, from porcine intestinal mucosa) were from Sigma. Synthetic peptides GRGDSP and GRGESP were purchased from Life Technologies, Inc. Function-blocking mAbs against various integrins were purchased from Chemicon, Inc. (Temecula, CA), including JB1A (anti-β1), FB12 (anti-α1), P1E6 (anti-α2), P1B5 (anti-α3), P1H4 (anti-α4), JBS5 (anti-α5β1), GoH3 (anti-α6), and LM609 (anti-αvβ3). For immunofluorescence microscopy studies, mAbs against talin (clone 8d4) and paxillin (clone 349) were obtained from Transduction Laboratories (San Diego, CA); mAbs against phosphotyrosine (clone PY-20) and human β1-integrin (clone DE9) were from Upstate Biotechnology Inc. (Lake Placid, NY); and mAb against human integrin α6-subunit (clone 4F10) was from Chemicon. mAbs against human MMP-1 (clone 41-1ES), MMP-2 (clone 42-5D11), and MMP-3 (clone 55-2A4) were from Oncogene Research Products (Cambridge, MA). Anti-Rac mAb (cone 23A8), anti-FAK mAb (clone 2A7), and anti-phosphotyrosine mAb used in immunoblotting studies (clone 4G10) were obtained from Upstate Biotechnology, Inc. Immunofluorescence microscopy was carried out as described (30Turner C.E. Glenney J.R.J. Burridge K. J. Cell Biol. 1990; 111: 1059-1068Crossref PubMed Scopus (525) Google Scholar). Cells adhered to coverslips were incubated with monoclonal antibodies against phosphotyrosine (clone PY-20), anti-α6 integrin (4F10), anti-β1integrin (clone DE9), anti-talin (clone 8d4), or anti-paxillin (clone 349) at 10 μg/ml, 50 μl/slip, and incubated at 37 °C for 1 h. Samples were washed four times with PBS plus 0.5% BSA and then incubated with fluorescein-conjugated horse anti-mouse IgG (20 μg/ml) at 37 °C for 30 min. To visualize actin cytoskeleton, cells were stained with rhodamine-conjugated phalloidin (Sigma) at 10 μg/ml, 100 μl per slip, and washed four times with PBS plus 0.5% BSA after incubation at room temperature for 1 h. For studies on intracellular signaling (see Figs. 5 and 6), 100-mm nontissue culture plastic dishes were precoated with various proteins at 10 μg/ml, 4 ml per dish, at 4 °C for 16 h, followed by blocking with 1% BSA at room temperature for 1 h. Cells were serum-starved and collected as described above. Protein-coated 100-mm dishes were kept at 37 °C and cells were plated at 2–3 ×106 cells/dish in 4 ml of 0.5% BSA-IMDM. Dishes and media were prewarmed at 37 °C, which helped to ensure consistent results with respect to cell adhesion and spreading within 5–15 min after plating.Figure 6Cyr61 and CTGF activate the small GTPase Rac in human skin fibroblasts. 1064SK fibroblasts were serum-starved, collected, and resuspended in serum-free IMDM and plated at 2–3 × 106 cells/100-mm dish precoated with Cyr61 (10 μg/ml) or CTGF (10 μg/ml) for various times as indicated. The dishes were prewarmed to 37 °C immediately before use. Time 0 indicates cells kept in suspension. Adherent cells were lysed at the indicated times, and activated Rac was affinity-precipitated and then immunoblotted with mAb against Rac protein. 50 μl of the clarified lysate from each sample was reserved for immunoblotting of total Rac protein. Data shown are representative of two independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) After incubation, cells were washed and lysed in a buffer (50 mm Tris·Cl, pH 7.5, 135 mm NaCl, 1% Triton X-100, 0.1% sodium deoxycholate, 2 mm EDTA, 50 mm NaF, 2 mm sodium orthovanadate, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 1 mm phenylmethylsulfonyl fluoride); immunoprecipitation and immunoblotting were carried out according to standard protocols (31Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor, New York1988: 488-510Google Scholar). To determine total FAK or paxillin in each sample, blots were stripped in a buffer (62.5 mm Tris-HCl, pH 6.8, 2% SDS, 100 mmβ-mercaptoethanol) at 60 °C and then reprobed with mAbs against FAK or paxillin. To study protein secreted by cells adhered on various substrates (as in Fig. 8), the conditioned media were collected after 24 h of cell incubation. The media were first centrifuged to remove cellular debris. Protein in the media was concentrated using Centricon YM-10 (molecular mass cut-off 10 kDa). An equal amount of conditioned media was loaded on SDS-PAGE and analyzed by immunoblotting with monoclonal antibodies against human MMP-1 (clone 41–1ES), MMP-2 (clone 42–5D11), and MMP-3 (clone 55–2A4). 1064SK fibroblasts were serum-starved for 24 h, harvested, and plated on 35-mm dishes precoated with proteins as described above. Total cell lysates were prepared and applied on SDS-PAGE, and immunoblotting was carried out using rabbit polyclonal antibodies against the dually phosphorylated active form p42/p44 MAPK (Thr(P)183/Tyr(P)185) at a 1:5000 dilution as suggested by the manufacturer (Promega, Madison, WI). 1064SK fibroblasts were serum-starved for 24 h, harvested, and plated on plastic dishes precoated with various proteins as described above. The Rac activation assay was done using Rac activation kit according to the manufacturer's protocol Biotechnology, 1064SK fibroblasts were serum-starved 24 h, and on protein-coated 100-mm plastic dishes in serum-free IMDM as described above. After incubation at 37 °C for various times, total cellular was and to using various R. J.A. K. in New Scholar). MMP-1 and MMP-2 were obtained from the American Type Culture MMP-3 was using with a to human MMP-3 and The blots were washed at 0.1% at and analyzed by a We showed that adhesion of primary human skin fibroblasts to Cyr61 is mediated through both integrin α6β1 and cell surface HSPGs (27Chen N. Chen C.C. Lau L.F. J. Biol. Chem. 2000; 275: 24953-24961Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). that CTGF is also a heparin-binding protein with sequence with we that CTGF may also fibroblast adhesion through a similar To test this we prepared microtiter wells coated with purified CTGF which primary human skin fibroblasts were allowed to adhere serum-free adhesion to CTGF was and and was by the of 2 μg/ml heparin in the medium These results that of the CTGF heparin-binding by heparin may with cell surface HSPGs, cell To test this we cultured human fibroblasts in the of sodium an of to sulfation of proteoglycans (29Rapraeger A.C. Krufka A. Olwin B.B. Science. 1991; 252: 1705-1708Crossref PubMed Scopus (1290) Google Scholar). to CTGF was this whereas adhesion of the same cells to and was The of sodium chlorate on cell adhesion to CTGF was by the of 10 in the culture that this is mediated through a sulfation (29Rapraeger A.C. Krufka A. Olwin B.B. Science. 1991; 252: 1705-1708Crossref PubMed Scopus (1290) Google Scholar). To the that cell surface proteoglycans are for cell adhesion to fibroblasts were with heparinase an that heparan sulfate proteoglycans J. R. R.O. J. Cell Biol. 107: PubMed Scopus Google Scholar, K. H. H. H. P. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). cells were to adhere to whereas the same cells adhered to or 1 of fibroblasts with chondroitinase ABC These results show that cell surface HSPGs to mediate adhesion of fibroblasts. adhesion to CTGF was by the of EDTA or in the assay not by In cell adhesion to type I collagen showed the same as to whereas cell adhesion to was not by as (27Chen N. Chen C.C. Lau L.F. J. Biol. Chem. 2000; 275: 24953-24961Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). This of fibroblasts adhesion to CTGF is consistent with the of an integrin To the specific integrin we the of The GRGDSP was to fibroblast adhesion to CTGF present at 2 a that cell adhesion to either or vitronectin, ligands of the integrin and the 2 The GRGESP was to cell adhesion to any Thus, adhesion of fibroblasts to CTGF is to be mediated through the integrins in these the and integrin To the specific integrin that mediates cell adhesion to we the of a of mAbs to of cells with mAbs against integrin or or the integrins and αVβ3 on fibroblast adhesion to CTGF not By contrast, mAb against integrin cell adhesion to whereas adhesion to was not 2 to laminin, a for integrin was This is skin fibroblasts as the receptor for K. K. T. K. Exp. Cell Res. PubMed Scopus Google Scholar). mAb against the integrin cell adhesion to CTGF not to was in adhesion to type I collagen, a for Together, these results show that adhesion of human skin fibroblasts to adhesion to both integrin α6β1 and cell surface of integrins and their adhesive ligands to intracellular resulting in a of cellular responses including focal adhesion and cell spreading E. Science. 1999; PubMed Scopus Google Scholar, P. K. M. J. M. M. K. G. J. Annu. Rev. Cell Dev. Biol. 1995; 11: PubMed Scopus Google Scholar). adhered to matrix proteins such as or are to form lamellipodia, and of cell although these within 30 min after both in by and in L. R.O. Mol. Biol. Cell. 1999; 10: PubMed Scopus Google Scholar). To understand the signaling responses as cells adhere to Cyr61 and we have the morphological changes and signaling responses in cells adhered to these proteins. of cells to and on coverslips coated with Cyr61 or CTGF within 10 min after as was the cells were allowed to adhere to or laminin, whereas cells plated on coverslips coated with BSA after 1 h of incubation Adherent fibroblasts were fixed with and stained with rhodamine-conjugated phalloidin to their actin microscopy showed that 30 min after fibroblasts adhered to Cyr61 or CTGF of actin the and of actin from the cell surface in of filopodia and A.F. Cell. 1996; 84: Full Text Full Text PDF PubMed Scopus Google Scholar, Cell. 1996; 84: Full Text Full Text PDF PubMed Scopus Google Scholar). These more at times, and by 60 min after cells to their In contrast, filopodia and in fibroblasts plated on or are in number and more in