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• W2014185712 abstract "Connective tissue growth factor (CCN2, also known as CTGF) is a matricellular protein that appears to play an important role in hepatic stellate cell (HSC)-mediated fibrogenesis. After signal peptide cleavage, the full-length CCN2 molecule comprises four structural modules (CCN21–4) and is susceptible to proteolysis by HSC yielding isoforms comprising essentially modules 3 and 4 (CCN23–4) or module 4 alone (CCN24). In this study we show that rat activated HSC are capable of adhesion to all three CCN2 isoforms via the binding of module 4 to integrin αvβ3, a process that is dependent on interactions between module 4 and cell surface heparan sulfate proteoglycans (HSPGs). These findings are based on several lines of evidence. First, integrin αvβ3 was detected in HSC lysates by immunoprecipitation and Western blot, and CCN24-mediated HSC adhesion was blocked by anti-integrin αvβ3 antibody. Second, as assessed by immunoprecipitation and solid phase binding assay, CCN24 bound directly to integrin αvβ3 in cell-free systems. Third, destruction or inhibition of synthesis of cell surface HSPGs with, respectively, heparinase or sodium chlorate abrogated HSC adhesion to CCN24. Fourth, prior occupancy of heparin-binding sites on CCN24 with soluble heparin completely blocked HSC adhesion. These findings indicate that integrin αvβ3 functions as a co-receptor with HSPGs for CCN24-mediated HSC adhesion. Furthermore, by peptide mapping and site-directed mutagenesis we demonstrated that the sequence IRTPKISKPIKFELSG within CCN24 is a unique binding domain for integrin αvβ3 that is sufficient to mediate integrin αvβ3- and HSPG-dependent HSC adhesion. These findings offer the possibility of developing novel antifibrotic therapies that target the integrin-binding domain. Connective tissue growth factor (CCN2, also known as CTGF) is a matricellular protein that appears to play an important role in hepatic stellate cell (HSC)-mediated fibrogenesis. After signal peptide cleavage, the full-length CCN2 molecule comprises four structural modules (CCN21–4) and is susceptible to proteolysis by HSC yielding isoforms comprising essentially modules 3 and 4 (CCN23–4) or module 4 alone (CCN24). In this study we show that rat activated HSC are capable of adhesion to all three CCN2 isoforms via the binding of module 4 to integrin αvβ3, a process that is dependent on interactions between module 4 and cell surface heparan sulfate proteoglycans (HSPGs). These findings are based on several lines of evidence. First, integrin αvβ3 was detected in HSC lysates by immunoprecipitation and Western blot, and CCN24-mediated HSC adhesion was blocked by anti-integrin αvβ3 antibody. Second, as assessed by immunoprecipitation and solid phase binding assay, CCN24 bound directly to integrin αvβ3 in cell-free systems. Third, destruction or inhibition of synthesis of cell surface HSPGs with, respectively, heparinase or sodium chlorate abrogated HSC adhesion to CCN24. Fourth, prior occupancy of heparin-binding sites on CCN24 with soluble heparin completely blocked HSC adhesion. These findings indicate that integrin αvβ3 functions as a co-receptor with HSPGs for CCN24-mediated HSC adhesion. Furthermore, by peptide mapping and site-directed mutagenesis we demonstrated that the sequence IRTPKISKPIKFELSG within CCN24 is a unique binding domain for integrin αvβ3 that is sufficient to mediate integrin αvβ3- and HSPG-dependent HSC adhesion. These findings offer the possibility of developing novel antifibrotic therapies that target the integrin-binding domain. Connective tissue growth factor (CCN2) is a cysteine-rich protein that stimulates a broad repertoire of cellular responses including proliferation, chemotaxis, adhesion, migration, and extracellular matrix production (1Brigstock D.R. Endocr. Rev. 1999; 20: 189-206Crossref PubMed Scopus (537) Google Scholar). CCN2 has been implicated in regulating diverse processes in vivo including angiogenesis, placentation, embryogenesis, differentiation, wound healing, and fibrosis, and its target cells include fibroblasts, endothelial cells, smooth muscle cells, epithelial cells, and neuronal cells (1Brigstock D.R. Endocr. Rev. 1999; 20: 189-206Crossref PubMed Scopus (537) Google Scholar, 2Moussad E.E. Brigstock D.R. Mol. Genet. Metab. 2000; 71: 276-292Crossref PubMed Scopus (441) Google Scholar, 3Lau L.F. Lam S.C. Exp. Cell Res. 1999; 248: 44-57Crossref PubMed Scopus (578) Google Scholar). The CCN2 primary translational product comprises 349 residues that are organized into a signal peptide and four structural modules that resemble an insulin-like growth factor-binding domain (module 1), a von Willebrand factor type C repeat (module 2), a thrombospondin type I repeat (module 3), and a C-terminal domain that contains a putative cysteine knot (module 4). The modular structure of CCN2 (CTGF) is conserved in other members of the CCN family that also includes CCN1 (CYR61), CCN3 (NOV), CCN4 (WISP-1), CCN5 (WISP-2), and CCN6 (WISP-3) (1Brigstock D.R. Endocr. Rev. 1999; 20: 189-206Crossref PubMed Scopus (537) Google Scholar, 3Lau L.F. Lam S.C. Exp. Cell Res. 1999; 248: 44-57Crossref PubMed Scopus (578) Google Scholar, 4Brigstock D.R. Goldschmeding R. Katsube K.I. Lam S.C. Lau L.F. Lyons K. Naus C. Perbal B. Riser B. Takigawa M. Yeger H. Mol. Pathol. 2003; 56: 127-128Crossref PubMed Scopus (203) Google Scholar). Studies of the binding properties of the individual modules within CCN proteins, together with their biological properties, have led to the suggestion that they act as matricellular proteins (3Lau L.F. Lam S.C. Exp. Cell Res. 1999; 248: 44-57Crossref PubMed Scopus (578) Google Scholar, 5Perbal B. Brigstock D.R. Lau L.F. Mol. Pathol. 2003; 56: 80-85Crossref PubMed Scopus (49) Google Scholar), a term originally used to describe a group of unrelated molecules (TSP1, TSP2, SPARC, tenascin C, and osteopontin) that interact contextually with specific cell surface receptors, cytokines, growth factors, and proteases to influence cell function by modulating cell-matrix interactions (6Bornstein P. J. Cell Biol. 1995; 130: 503-506Crossref PubMed Scopus (577) Google Scholar, 7Bornstein P. Matrix Biol. 2000; 19: 555-556Crossref PubMed Scopus (64) Google Scholar, 8Bornstein P. Sage E.H. Curr. Opin. Cell Biol. 2002; 14: 608-616Crossref PubMed Scopus (754) Google Scholar).Recent reports have provided strong evidence of a role for CCN2 in liver fibrosis, especially as a mediator of transforming growth factor-β action. Although several cell types produce CCN2 in fibrotic liver (9Abou-Shady M. Friess H. Zimmermann A. di Mola F.F. Guo X.Z. Baer H.U. Buchler M.W. Liver. 2000; 20: 296-304Crossref PubMed Scopus (105) Google Scholar, 10Williams E.J. Gaca M.D. Brigstock D.R. Arthur M.J. Benyon R.C. J. Hepatol. 2000; 32: 754-761Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 11Paradis V. Dargere D. Vidaud M. De Gouville A.C. Huet S. Martinez V. Gauthier J.M. Ba N. Sobesky R. Ratziu V. Bedossa P. Hepatology. 1999; 30: 968-976Crossref PubMed Scopus (304) Google Scholar), attention has become focused on hepatic stellate cells (HSC), 1The abbreviations used are: HSC, hepatic stellate cell(s); BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent assay; HSPGs, heparan sulfate proteoglycans; LN, laminin; VN, vitronectin; MBP, maltose-binding fusion protein; PBS, phosphate-buffered saline.1The abbreviations used are: HSC, hepatic stellate cell(s); BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent assay; HSPGs, heparan sulfate proteoglycans; LN, laminin; VN, vitronectin; MBP, maltose-binding fusion protein; PBS, phosphate-buffered saline. the liver-specific pericytes located in the space of Disse that lie in close contact with hepatocytes and sinusoidal endothelial cells. Following liver injury, HSC undergo a transition from quiescent vitamin A-rich cells to activated vitamin A-deficient, proliferative, fibrogenic, and contractile myofibroblasts. CCN2 expression by HSC is enhanced during the process of activation as well as by transforming growth factor-β, vascular endothelial growth factor, platelet-derived growth factor, lipid peroxidation products, and acetaldehyde (10Williams E.J. Gaca M.D. Brigstock D.R. Arthur M.J. Benyon R.C. J. Hepatol. 2000; 32: 754-761Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 12Paradis V. Dargere D. Bonvoust F. Vidaud M. Segarini P. Bedossa P. Lab. Invest. 2002; 82: 767-774Crossref PubMed Google Scholar). In response to exogenous CCN2, HSC demonstrate increased migration, proliferation, adhesion, and expression of type I collagen (12Paradis V. Dargere D. Bonvoust F. Vidaud M. Segarini P. Bedossa P. Lab. Invest. 2002; 82: 767-774Crossref PubMed Google Scholar, 13Rachfal A.W. Brigstock D.R. Hepatol. Res. 2003; 26: 1-9Crossref PubMed Scopus (213) Google Scholar, 14Ball D.K. Rachfal A.W. Kemper S.A. Brigstock D.R. J. Endocrinol. 2003; 176: R1-R7Crossref PubMed Scopus (79) Google Scholar).Over the last few years, progress has begun to be made in determining the mechanisms that underlie the diverse effect of CCN proteins on cell function. CCN proteins have emerged as novel ligands for integrins and can functionally engage different integrin subtypes to elicit specific biological effects. Integrins are composed of α and β subunits, and each α-β combination has its own binding specificity and signaling properties that can impact cell behavior and cell-matrix or cell-cell interactions (15Hynes R.O. Cell. 2002; 110: 673-687Abstract Full Text Full Text PDF PubMed Scopus (6764) Google Scholar, 16Giancotti F.G. Ruoslahti E. Science. 1999; 285: 1028-1033Crossref PubMed Scopus (3785) Google Scholar). Because integrins can transduce extracellular binding events into intracellular signaling cascades (17Chen C.C. Chen N. Lau L.F. J. Biol. Chem. 2001; 276: 10443-10452Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar, 18Chen C.C. Mo F.E. Lau L.F. J. Biol. Chem. 2001; 276: 47329-47337Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar), the ability of CCN proteins to interact with different integrins provides a mechanistic basis for their broad ranging biological activities (3Lau L.F. Lam S.C. Exp. Cell Res. 1999; 248: 44-57Crossref PubMed Scopus (578) Google Scholar). To date, the most compelling data have emerged for CCN1 that uses different integrin subtypes to individually regulate adhesion, migration, and proliferation in fibroblasts, endothelial cells, smooth muscle cells, or monocytes (17Chen C.C. Chen N. Lau L.F. J. Biol. Chem. 2001; 276: 10443-10452Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar, 19Chen N. Chen C.C. Lau L.F. J. Biol. Chem. 2000; 275: 24953-24961Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 20Grzeszkiewicz T.M. Kirschling D.J. Chen N. Lau L.F. J. Biol. Chem. 2001; 276: 21943-21950Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 21Leu S.J. Liu Y. Chen N. Chen C.C. Lam S.C. Lau L.F. J. Biol. Chem. 2003; 278: 33801-33808Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 22Babic A.M. Chen C.C. Lau L.F. Mol. Cell. Biol. 1999; 19: 2958-2966Crossref PubMed Google Scholar, 23Schober J.M. Chen N. Grzeszkiewicz T.M. Jovanovic I. Emeson E.E. Ugarova T.P. Ye R.D. Lau L.F. Lam S.C. Blood. 2002; 99: 4457-4465Crossref PubMed Scopus (209) Google Scholar, 24Grzeszkiewicz T.M. Lindner V. Chen N. Lam S.C. Lau L.F. Endocrinology. 2002; 143: 1441-1450Crossref PubMed Scopus (133) Google Scholar).Although CCN2 likely functions as a paracrine and autocrine fibrogenic factor for HSC, there is no information regarding the nature of cell surface CCN2 receptors on HSC. In the present study we show that integrin αvβ3 and cell surface heparan sulfate proteoglycans (HSPGs) are indispensable for adhesion of HSC to CCN2. The integrin αvβ3 binding property localizes to a unique non-RGD region in module 4 (residues 257–272) that supports HSC adhesion via integrin αvβ3- and HSPG-dependent mechanisms.MATERIALS AND METHODSAntibodies, Peptides, and Reagents—Function-blocking monoclonal anti-human anti-αvβ3 (LM609), anti-αv (LM142), and anti-β3 (B3A) as well as purified human integrin αvβ3 were purchased from Chemicon, Inc. (Temicula, CA). Heparinase I, chondroitinase ABC, bovine serum albumin (BSA; protease-free and γ-globulin-free), mouse normal IgG, laminin (LN), vitronectin (VN), echistatin, and sodium chlorate (NaClO3) were obtained from Sigma. Synthetic peptides GRGDsp, GRGEsp, and Dulbecco’s modified Eagle’s medium were purchased from Invitrogen. Heparin was from USB (Cleveland, OH). The Cy-QUANT® cell proliferation assay kit was from Molecular Probes (Eugene, OR). Protein A-Sepharose beads were from Pierce, and MBP was from New England BioLabs (Beverley, MA).CCN2 Proteins and Peptides—Recombinant human CCN2 was produced and purified as described (14Ball D.K. Rachfal A.W. Kemper S.A. Brigstock D.R. J. Endocrinol. 2003; 176: R1-R7Crossref PubMed Scopus (79) Google Scholar, 25Ball D.K. Moussad E.E. Rageh M.A. Kemper S.A. Brigstock D.R. Reproduction. 2003; 125: 271-284Crossref PubMed Scopus (46) Google Scholar). CCN2 proteins used in this study were intact 38-kDa CCN2 containing all four modules (CCN21–4), 16–20-kDa CCN2 containing only modules 3 and 4 (CCN23–4), and a fusion protein (MBP) containing 10-kDa CCN2 that comprised essentially module 4 alone (residues 247–349; CCN24) (14Ball D.K. Rachfal A.W. Kemper S.A. Brigstock D.R. J. Endocrinol. 2003; 176: R1-R7Crossref PubMed Scopus (79) Google Scholar). The structures of these CCN2 isoforms are shown in Fig. 1. CCN24 mutant proteins were generated by polymerase chain reaction of human CCN24 cDNA in pMAL-C2 (14Ball D.K. Rachfal A.W. Kemper S.A. Brigstock D.R. J. Endocrinol. 2003; 176: R1-R7Crossref PubMed Scopus (79) Google Scholar) using a QuikChange® II site-directed mutagenesis kit (Stratagene, La Jolla, CA). The primers were designed to introduce alanine substitutions into four adjacent residues over residues 257–272 as follows: 5′-AGTGCGCCGCTGCTGCCAAAATCTCCAAGCCTATCAAGTTTGAGCTTTCTGGCTGCACC-3′ to generate I257A/R258A/T259A/P260A (M1); 5′-GCATCCGTACTCCCGCGCGCCGCGCCTATCAAGTTTGAG-3′ to generate K261A/I262A/S263A/K264A (M2); 5′-CCCAAAATCTCCAAGGCTGCCGCGGCTGAGCTTTCTGGC-3′ to generate P265A/I266A/K267A/F268A (M3); and 5′-GCCTATCAAGTTTGCGGCTGCTGCCTGCACCAGCATG-3′ to generate E269A/L270A/S271A/G272A (M4). The various mutants are shown in Fig. 1. The mutant sequences were verified by DNA sequencing, and the proteins were produced and purified by sequential amylose affinity chromatography and heparin affinity chromatography as described (14Ball D.K. Rachfal A.W. Kemper S.A. Brigstock D.R. J. Endocrinol. 2003; 176: R1-R7Crossref PubMed Scopus (79) Google Scholar).Ten synthetic peptides spanning the entire 103 C-terminal residues of CCN2 were synthesized by Mimotopes Inc. (Clayton, Australia) (Table I). All peptides were synthesized with acetylated N termini and amidated C termini except CCN24-(247–260), which was synthesized with a free N-terminal amine, and CCN24-(326–349), which was synthesized with an acid C terminus. Cys292 in CCN24-(285–292) was replaced with Ser to prevent an intrachain disulfide bridge to Cys287.Table IPeptide sequences within CCN24PeptideSequenceResiduesP1EENIKKGKKCIRTaFree N-terminal amine.247-260P2IRTPKISKPIKFELSG257-272P3TPKISKPIKFELSG259-272P4CTSMKTYRAKFCGV273-286P5GVCTDGRS292285-292P6CTPHRTTTLPVEFK293-306P7FKCPDGEVMKKNMMFIKT305-322P8MFIKTCA318-324P9ACHYN324-328P10HYNCPGDNDIFESLYYRKMYGDMAbAcid C terminus.326-349a Free N-terminal amine.b Acid C terminus. Open table in a new tab HSC Isolation and Culture—HSC were isolated from normal male Sprague-Dawley rats by sequential perfusion with Pronase/collagenase B and purification by density gradient separation (26Vyas S.K. Leyland H. Gentry J. Arthur M.J. Gastroenterology. 1995; 109: 889-898Abstract Full Text PDF PubMed Scopus (77) Google Scholar). The cells were cultured on plastic culture dishes in Dulbecco’s modified Eagle’s medium supplemented with 25 mm Hepes buffer and 10% fetal bovine serum. The adhesion assays were performed between the first and third serial passages on three different HSC preparations that had been allowed to autonomously activate in culture.Cell Adhesion Assay—HSC were detached using 1 mm EDTA in PBS (137 mm NaCl, 2.7 mm KCl, 4.3 mm Na2HPO4, 1.4 mm KH2PO4, pH 7.3), washed twice in Dulbecco’s modified Eagle’s medium, and resuspended in serum-free medium containing 0.5% BSA. CCN2 proteins or peptides or control cell adhesion molecules were diluted to the desired concentration in PBS and used at 50 μl/well to precoat 96-well plates (Costar, Corning, NY) at 4 °C for 16 h. The wells were then blocked for 1 h with PBS containing 1% BSA prior to addition of 50 μl of HSC suspension (2.5 × 105 cells/ml) for 20 min at 37 °C. The wells were washed three times with PBS, and adherent cells were fixed with 10% formalin and stained by the addition of 100 μl of CyQUANT GR dye/cell lysis buffer to each sample well. After incubation in the dark for 5 min at room temperature, adherent cells were quantitated by measurement of the sample fluorescence intensity using a micro-plate reader (CytoFluor™ 2350) at an excitation of 485 nm and an emission of 530 nm.Immunoprecipitation and Immunoblotting—1 × 106 activated HSC were plated in 10-cm culture dishes and incubated for 24 h. The cells were washed with cold PBS prior to addition of 1 ml of Nonidet P-40 buffer (20 mm Tris-HCl, pH 7.4, containing 1% Nonidet P-40, 150 mm NaCl, 1 mm MgCl2, 1 mm CaCl2, 10% glycerol, 1 mm Na3VO4, 1 mm 4-(2-aminoethyl)benzene-sulfonyl fluoride, 1 μg/ml leupeptin, and 1 μg/ml aprotinin) with rocking at 4 °C for 1 h. The cell lysates were collected and centrifuged for 5 min at 15,000 × g. The supernatants were incubated with anti-integrin αv (1:200) at 4 °C for 16 h, and the immune complexes were precipitated with 25 μl of protein A-Sepharose beads for 1 h. The beads were washed three times with 1 ml of cold Nonidet P-40 buffer and then boiled for 10 min in sample buffer. The samples were separated on 8% SDS-polyacrylamide gels and transferred onto nitrocellulose. After blocking the membrane with 5% nonfat milk, the filter was incubated with anti-integrin β3 (1:1000) for 1 h. The membrane was then incubated for 1 h with horseradish peroxidase-linked anti-mouse IgG (1:4000) diluted in TBS/T (10 mm Tris-HCl, pH 8.0, containing 150 mm NaCl and 0.1% Tween 20) and washed extensively with TBS/T before detection using the ECL system.CCN2 Isoforms and Integrin αvβ3 Binding Assay—2 μg of human integrin αvβ3 were incubated with rocking at 4 °C for 2 h with 4 μg of CCN21–4, 4 μg of CCN23–4, or 10 μg of CCN24 in 1 ml of Nonidet P-40 buffer. The mixtures were then incubated at 4 °C for 16 h with 1:100 dilution of an immunoprecipitating polyclonal rabbit anti-CCN2 antibody (previously shown to detect all CCN2 isoforms; see Ref. 25Ball D.K. Moussad E.E. Rageh M.A. Kemper S.A. Brigstock D.R. Reproduction. 2003; 125: 271-284Crossref PubMed Scopus (46) Google Scholar) or 10 μg of mouse IgG followed by the addition of 25 μl of protein A-Sepharose beads for 1 h. In inhibition assays, 2 μg of human integrin αvβ3 were preincubated for 1 h with 20 μm echistatin in 1 ml of Nonidet P-40 buffer prior to the addition of 10 μg of CCN24. The samples were separated on 8% SDS-polyacrylamide gels and transferred onto a nitro-cellulose membrane that was incubated with anti-human αvβ3 (1:1000) diluted in TBS/T and developed as described above.Solid Phase Binding Assay—The binding of integrin αvβ3 to immobilized CCN2 isoforms was measured as follows. Microtiter wells (Dynex Technology, Chantilly, VA) were precoated with different CCN2 isoforms at the desired concentrations for 16 h at 4 °C and then blocked at room temperature for 2 h with PBS containing 2% BSA, 1 mm CaCl2, and 1 mm MgCl2. The plate was washed four times with PBS and then incubated at room temperature for 3 h with 1 μg/ml integrin αvβ3 in blocking solution. In inhibition studies, integrin αvβ3 was preincubated with 10 μm individual CCN24 synthetic peptides for 1 h. The plate was developed by the addition of anti-human αvβ3 monoclonal antibody diluted in blocking solution (1:1000) followed by horseradish peroxidase-conjugated goat anti-mouse IgG (1:4000). The color reaction was developed using horseradish peroxidase ELISA reagents (Chemicon, Temecula, CA), and the absorbance at 450 nm was measured using a HTS700 Bio Assay Reader (PerkinElmer Life Sciences).RESULTSInvolvement of Integrins in Adhesion of Activated HSC to CCN2 Isoforms—It has previously been shown that cultured HSC produce CCN2 and its low mass isoforms as a function of activation in culture (10Williams E.J. Gaca M.D. Brigstock D.R. Arthur M.J. Benyon R.C. J. Hepatol. 2000; 32: 754-761Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar) and that CCN2 stimulates HSC proliferation and extracellular matrix production in vitro (12Paradis V. Dargere D. Bonvoust F. Vidaud M. Segarini P. Bedossa P. Lab. Invest. 2002; 82: 767-774Crossref PubMed Google Scholar). In view of the emerging role of CCN proteins as cell adhesion molecules (3Lau L.F. Lam S.C. Exp. Cell Res. 1999; 248: 44-57Crossref PubMed Scopus (578) Google Scholar), we examined the ability of CCN2 to support the adhesion of activated HSC. As previously shown (27Gao R. Brigstock D. Hepatol. Res. 2003; 27: 214-220Crossref PubMed Scopus (109) Google Scholar), all three CCN2 isoforms promoted dose-dependent HSC adhesion over a comparable concentration range (0.5–2 μg/ml), with maximal adhesion elicited by 2 μg/ml. This effect was comparable to the well characterized cell adhesion molecules, VN and LN (Fig. 2A and Ref. 27Gao R. Brigstock D. Hepatol. Res. 2003; 27: 214-220Crossref PubMed Scopus (109) Google Scholar).Fig. 2Regulation of adhesion of HSC to all CCN2 isoforms by divalent cations and RGD peptides.A, microtiter wells were precoated at 4 °C for 16 h with CCN21–4 (2 μg/ml), CCN23–4 (2 μg/ml), CCN24 (8 μg/ml), LN (1 μg/ml), or VN (4 μg/ml) and then blocked with PBS, 1% BSA for 1 h. Rat activated HSC (2.5 × 105/ml) were preincubated in serum-free medium for 30 min in vehicle buffer (no Add) or EDTA (10 mm) prior to the addition to individual wells at 50 μl/well. After incubation at 37 °C for 20 min, the adherent cells were washed, fixed, and stained with CyQUANT GR dye and quantified by measuring the fluorescence intensity at an excitation of 485 nm and an emission of 530 nm. B, HSC adhesion assays were performed on microtiter wells that had been precoated with MBP (8 μg/ml) or CCN24 (8 μg/ml) following preincubation of the cells for 30 min with EDTA (10 mm) or the addition of Ca2+ (10 mm), Mn2+ (10 mm), or Mg2+ (10 mm) either alone or in combination. C, HSC were preincubated with echistatin (2 μm), GRGDsp (1 mm), GRGEsp (1 mm), or vehicle buffer alone (No Add) for 30 min prior to adding HSC to the wells. D, microtiter wells were coated with CCN24 (8 μg/ml) or VN (4 μg/ml) prior to the addition of HSC that had been preincubated for 30 min with the indicated concentrations of echistatin. The data are the means ± S.D. of quadruplicate determinations and are representative of three experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To assess the possibility that CCN2-mediated HSC adhesion involved cell surface integrins, we examined the effect of divalent cations on cell adhesion. Following incubation of the cells with 10 mm EDTA, CCN21–4- and CCN23–4-mediated HSC adhesion was decreased by 50%, and CCN24-mediated HSC adhesion was inhibited by 90% (Fig. 2A). The inhibitory effect of EDTA on the ability of CCN24 to support HSC adhesion was restored by the addition of the divalent cations Ca2+, Mg2+, or Mn2+ (Fig. 2B). In the absence of EDTA, CCN24-mediated HSC adhesion was supported in the presence of Mg2+ or Mn2+ but completely abolished by Ca2+.Mn2+, but not Mg2+, was able to overcome the inhibitory effect of Ca2+ on HSC adhesion by CCN24 (Fig. 2B). The cation dependence of CCN2-mediated HSC adhesion was consistent with the possible involvement of integrins in this process.To further investigate the likely role of integrins, we took advantage of the fact that many integrin-ligand interactions are RGD-dependent and can be inhibited by RGD-containing peptides. As shown in Fig. 2C, the hexapeptide GRGDsp (1 mm) was able to block CCN21–4-, CCN23–4-, or CCN24-mediated cell adhesion by 45, 51, or 58%, respectively, whereas the control hexapeptide GRGEsp (1 mm) was ineffective. Additionally, echistatin, a RGD-containing disintegrin with a binding preference for β3 integrin that is at least 500 times more potent than short RGDX peptides (28Gould R.J. Polokoff M.A. Friedman P.A. Huang T.F. Holt J.C. Cook J.J. Niewiarowski S. Proc. Soc. Exp. Biol. Med. 1990; 195: 168-171Crossref PubMed Scopus (484) Google Scholar), inhibited CCN21–4-, CCN23–4-, or CCN24-mediated cell adhesion by 58, 62, and 69%, respectively, when tested at 2 μm. HSC were also shown to bind to VN, a RGD-containing ligand of integrin αvβ3, and this effect was completely inhibited by either GRGDsp or echistatin (Fig. 2C). Although the effect of echistatin on CCN24- or VN-mediated HSC adhesion was dose-dependent, the residual level of HSC adhesion in the presence of saturating concentrations of echistatin (2–3 μm) was higher when CCN24 was used as a substrate as compared with VN (Fig. 2D). These data were comparable to the greater GRGDsp sensitivity of HSC adhesion to VN as compared with CCN24 (Fig. 2C).Because integrin αvβ3 has been implicated in the adhesion of certain other cell types to CCN proteins (22Babic A.M. Chen C.C. Lau L.F. Mol. Cell. Biol. 1999; 19: 2958-2966Crossref PubMed Google Scholar, 29Ellis P.D. Metcalfe J.C. Hyvonen M. Kemp P.R. J. Vasc. Res. 2003; 40: 234-243Crossref PubMed Scopus (28) Google Scholar, 30Leu S.J. Lam S.C. Lau L.F. J. Biol. Chem. 2002; 277: 46248-46255Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar), we next investigated the production of integrin αvβ3 by activated HSC using sequential immunoprecipitation with anti-integrin αv antibody and immunoblotting with anti-integrin β3. As shown in Fig. 3A, a 105-kDa immunoreactive protein was detected on the Western blot, which corresponded to the predicted size of the β3 subunit and thus confirmed that integrin αvβ3 was produced by rat activated HSC. To further clarify a role of integrin αvβ3 in contributing to CCN24-HSC interactions, a cell adhesion assay was performed in the presence of a specific anti-integrin αvβ3 monoclonal antibody. As shown in Fig. 3B, the antibody inhibited adhesion of HSC to CCN24 by more than 60%, whereas mouse IgG had no inhibitory effect on CCN24-mediated cell adhesion. As expected, anti-αvβ3 completely blocked the cell adhesion to VN but had no effect on the adhesion of HSC to LN, the latter of which is not a ligand for integrin αvβ3.Fig. 3Production of integrin αvβ3by rat activated HSC and its involvement in CCN24-mediated cell adhesion.A, anti-integrin αv immunoprecipitates (IP) of lysates from activated HSC were subjected to SDS-PAGE and Western blot analysis using anti-integrin β3 antibody. The results are representative of three separate experiments. B, P2 (10 μm), anti-integrin αvβ3 (25 μg/ml), mouse IgG (25 μg/ml), or vehicle buffer alone (no add) were added to the cell suspension for 30 min at room temperature. The cells were plated into microtiter wells that had been precoated with MBP (8 μg/ml), CCN24 (8 μg/ml), LN (1 μg/ml), or VN (4 μg/ml). The data are the means ± S.D. of quadruplicate determinations and are representative of three experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Collectively, these data indicated that adhesion of HSC to CCN24 involves binding to integrin αvβ3. However, the inability of integrin αvβ3 antagonists to fully block CCN2-mediated cell adhesion (as compared with their full inhibition of VN-mediated cell adhesion) suggested that other mechanisms are also likely to contribute to the binding of HSC to CCN2.Binding of CCN2 to Integrin αvβ3 in Cell-free Systems—To further characterize the interaction between CCN2 and integrin αvβ3, the three CCN2 isoforms were individually mixed with purified integrin αvβ3 prior to immunoprecipitation with rabbit anti-CCN2 polyclonal antibody and immunoblotting with anti-integrin αvβ3. As shown in Fig. 4A, a direct binding between integrin αvβ3 and each CCN2 protein was demonstrated based on the ability to detect integrin αvβ3 on the Western blot. Additional studies with CCN24 showed that its binding to integrin αvβ3 was completely blocked by echistatin and that the immunoprecipitation occurred specifically in the presence of CCN2 antibodies and not a normal IgG control (Fig. 4A). To confirm the cell-free binding of integrin αvβ3 to CCN2 using an alternative approach, a solid phase binding assay was used in which each CCN2 protein or VN were individually coated onto microtiter wells that were subsequently incubated with purified integrin αvβ3. The presence of immobilized integrin αvβ3 was detected by ELISA using an anti-integrin αvβ3 antibody. As shown in Fig. 4B, each CCN2 isoform or VN was found to bind to integrin αvβ3, with the strongest signal exhibited by CCN24. This effect was confirmed in dose-response experiments (Fig. 4C) that showed a higher level of maximal binding of integrin αvβ3 to CCN24 (saturation at 8 μg/ml CCN24) as compared wi" @default.
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• W2014185712 title "Connective Tissue Growth Factor (CCN2) Induces Adhesion of Rat Activated Hepatic Stellate Cells by Binding of Its C-terminal Domain to Integrin αvβ3 and Heparan Sulfate Proteoglycan" @default.
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• W2014185712 doi "https://doi.org/10.1074/jbc.m313204200" @default.
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