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• W2087823284 abstract "CCN1 (cysteine-rich 61) and CCN2 (connective tissue growth factor) are growth factor-inducible immediate-early gene products found in atherosclerotic lesions, restenosed blood vessels, and healing cutaneous wounds. Both CCN proteins have been shown to support cell adhesion and induce cell migration through interaction with integrin receptors. Recently, we have identified integrin αMβ2 as the major adhesion receptor mediating monocyte adhesion to CCN1 and CCN2 and have shown that the αMI domain binds specifically to both proteins. In the present study, we demonstrated that activated monocytes adhered to a synthetic peptide (CCN1-H2, SSVKKYRPKYCGS) derived from a conserved region within the CCN1 C-terminal domain, and this process was blocked by the anti-αM monoclonal antibody 2LPM19c. Consistently, a glutathione S-transferase (GST) fusion protein containing the αMI domain (GST-αMI) bound to immobilized CCN1-H2 as well as to the corresponding H2 sequence in CCN2 (CCN2-H2, TSVKTYRAKFCGV). By contrast, a scrambled CCN1-H2 peptide and an 18-residue peptide derived from an adjacent sequence of CCN1-H2 failed to support monocyte adhesion or αMI domain binding. To confirm that the CCN1-H2 sequence within the CCN1 protein mediates αMβ2 interaction, we developed an anti-peptide antibody against CCN1-H2 and showed that it specifically blocked GST-αMI binding to intact CCN1. Collectively, these results identify the H2 sequence in CCN1 and CCN2 as a novel integrin αMβ2 binding motif that bears no apparent homology to any αMβ2 binding sequence reported to date. CCN1 (cysteine-rich 61) and CCN2 (connective tissue growth factor) are growth factor-inducible immediate-early gene products found in atherosclerotic lesions, restenosed blood vessels, and healing cutaneous wounds. Both CCN proteins have been shown to support cell adhesion and induce cell migration through interaction with integrin receptors. Recently, we have identified integrin αMβ2 as the major adhesion receptor mediating monocyte adhesion to CCN1 and CCN2 and have shown that the αMI domain binds specifically to both proteins. In the present study, we demonstrated that activated monocytes adhered to a synthetic peptide (CCN1-H2, SSVKKYRPKYCGS) derived from a conserved region within the CCN1 C-terminal domain, and this process was blocked by the anti-αM monoclonal antibody 2LPM19c. Consistently, a glutathione S-transferase (GST) fusion protein containing the αMI domain (GST-αMI) bound to immobilized CCN1-H2 as well as to the corresponding H2 sequence in CCN2 (CCN2-H2, TSVKTYRAKFCGV). By contrast, a scrambled CCN1-H2 peptide and an 18-residue peptide derived from an adjacent sequence of CCN1-H2 failed to support monocyte adhesion or αMI domain binding. To confirm that the CCN1-H2 sequence within the CCN1 protein mediates αMβ2 interaction, we developed an anti-peptide antibody against CCN1-H2 and showed that it specifically blocked GST-αMI binding to intact CCN1. Collectively, these results identify the H2 sequence in CCN1 and CCN2 as a novel integrin αMβ2 binding motif that bears no apparent homology to any αMβ2 binding sequence reported to date. CCN1 1The abbreviations used are: CCN, CYR61/CTGF/nephroblastoma-overexpressed; BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent assay; Fbg, fibrinogen; GST, glutathione S-transferase; HRP, horseradish peroxidase; HSPGs, heparan sulfate proteoglycans; PVA, polyvinyl alcohol; HRP, horseradish peroxidase; PEO, polyethylene oxide. (cysteine-rich 61, CYR61) and CCN2 (connective tissue growth factor, CTGF), products of immediate-early genes transcriptionally activated in fibroblasts and smooth muscle cells upon growth factor stimulation, belong to the CCN (CYR61/CTGF/nephroblastoma-overexpressed) family of matricellular signaling molecules that have been implicated in diverse biological functions (1Lau L.F. Lam S.C. Exp. Cell Res. 1999; 248: 44-57Crossref PubMed Scopus (581) Google Scholar, 2Brigstock D.R. Endocr. Rev. 1999; 20: 189-206Crossref PubMed Scopus (541) Google Scholar, 3Perbal B. Mol. Pathol. 2001; 54: 57-79Crossref PubMed Scopus (316) Google Scholar). Other CCN family members include CCN3 (nephroblastoma-overexpressed, Nov) and the wnt-1-induced secreted proteins CCN4 (WISP-1), CCN5 (WISP-2), and CCN6 (WISP-3). These conserved and modular proteins consist of an N-terminal secretory peptide followed by four structural domains that include 1) an insulin-like growth factor-binding protein homology domain, 2) a von Willebrand factor type C domain, 3) a thrombospondin type 1 repeat homology domain, and 4) a C-terminal domain with heparin binding motifs and sequence similarity to the C termini of von Willebrand factor and mucin (4Bork P. FEBS Lett. 1993; 327: 125-130Crossref PubMed Scopus (554) Google Scholar). Consistent with their localization to the extracellular matrix and their homology to extracellular matrix proteins, several CCN members have been shown to support cell adhesion, induce adhesive signaling, promote cell migration, and augment growth factor-induced cell proliferation through interaction with integrin receptors and cell surface heparan sulfate proteoglycans (HSPGs) (5Kireeva M.L. Lam S.C. Lau L.F. J. Biol. Chem. 1998; 273: 3090-3096Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 6Jedsadayanmata 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, 7Chen N. Chen C.C. Lau L.F. J. Biol. Chem. 2000; 275: 24953-24961Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 8Babic A.M. Kireeva M.L. Kolesnikova T.V. Lau L.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6355-6360Crossref PubMed Scopus (433) Google Scholar, 9Babic A.M. Chen C.C. Lau L.F. Mol. Cell. Biol. 1999; 19: 2958-2966Crossref PubMed Google Scholar, 10Grzeszkiewicz T.M. Kirschling D.J. Chen N. Lau L.F. J. Biol. Chem. 2001; 276: 21943-21950Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 11Chen C.C. Chen N. Lau L.F. J. Biol. Chem. 2001; 276: 10443-10452Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar, 12Schober 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 (213) Google Scholar, 13Grzeszkiewicz T.M. Lindner V. Chen N. Lam S.C. Lau L.F. Endocrinology. 2002; 143: 1441-1450Crossref PubMed Scopus (133) Google Scholar). Furthermore, both CCN1 and CCN2 induce neovascularization in the rat corneal micropocket assay (8Babic A.M. Kireeva M.L. Kolesnikova T.V. Lau L.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6355-6360Crossref PubMed Scopus (433) Google Scholar, 9Babic A.M. Chen C.C. Lau L.F. Mol. Cell. Biol. 1999; 19: 2958-2966Crossref PubMed Google Scholar). Recently, we showed that proangiogenic activities of CCN1 are mediated through integrins α6β1 and αvβ3 in unactivated and activated human umbilical vein endothelial cells, respectively (14Leu S.J. Lam S.C. Lau L.F. J. Biol. Chem. 2002; 277: 46248-46255Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar). The expression of CCN1 is essential for normal embryonic development inasmuch as targeted disruption of the CCN1 gene in mouse results in embryonic lethality (15MO F.E. Muntean A.G. Chen C.C. Stolz D.B. Watkins S.C. Lau L.F. Mol. Cell. Biol. 2002; 22: 8709-8720Crossref PubMed Scopus (346) Google Scholar). Interestingly, the majority of the CCN1-null embryos exhibit vascular defects, consistent with an essential role of CCN1 in normal embryonic angiogenesis. In adults, CCN1 is present at low levels in the cardiovascular system (16Kireeva 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, 17Hilfiker A. Hilfiker-Kleiner D. Fuchs M. Kaminski K. Lichtenberg A. Rothkotter H.J. Schieffer B. Drexler H. Circulation. 2002; 106: 254-260Crossref PubMed Scopus (90) Google Scholar); however, its expression is up-regulated in a number of vascular diseases including atherosclerosis and proliferative restenosis (12Schober 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 (213) Google Scholar, 13Grzeszkiewicz T.M. Lindner V. Chen N. Lam S.C. Lau L.F. Endocrinology. 2002; 143: 1441-1450Crossref PubMed Scopus (133) Google Scholar, 17Hilfiker A. Hilfiker-Kleiner D. Fuchs M. Kaminski K. Lichtenberg A. Rothkotter H.J. Schieffer B. Drexler H. Circulation. 2002; 106: 254-260Crossref PubMed Scopus (90) Google Scholar, 18Wu K.J. Yee A. Zhu N.L. Gordon E.M. Hall F.L. Int. J. Mol. Med. 2000; 6: 433-440Crossref PubMed Google Scholar). High expression of CCN2 in human advanced atherosclerotic lesions has also been observed (19Oemar B.S. Werner A. Garnier J.M. Do D.D. Godoy N. Nauck M. Marz W. Rupp J. Pech M. Luscher T.F. Circulation. 1997; 95: 831-839Crossref PubMed Scopus (290) Google Scholar, 20Oemar B.S. Luscher T.F. Arterioscler. Thromb. Vasc. Biol. 1997; 17: 1483-1489Crossref PubMed Scopus (149) Google Scholar). Moreover, both CCN1 and CCN2 are expressed during cutaneous wound healing (21Latinkic B.V. MO F.E. Greenspan J.A. Copeland N.G. Gilbert D.J. Jenkins N.A. Ross S.R. Lau L.F. Endocrinology. 2001; 142: 2549-2557Crossref PubMed Scopus (47) Google Scholar, 22Igarashi A. Okochi H. Bradham D.M. Grotendorst G.R. Mol. Biol. Cell. 1993; 4: 637-645Crossref PubMed Scopus (645) Google Scholar, 23Chen C.C. MO F.E. Lau L.F. J. Biol. Chem. 2001; 276: 47329-47337Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar). It is well established that leukocyte adhesion and emigration play an important role in inflammation, wound healing, and atherosclerosis (24Springer T.A. Cell. 1994; 76: 301-314Abstract Full Text PDF PubMed Scopus (6409) Google Scholar). In a recent report, we showed that human peripheral blood monocytes adhere to CCN1 and CCN2 in an activation-dependent manner through integrin αMβ2 (12Schober 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 (213) Google Scholar), underscoring the importance of these proteins in the pathophysiologic function of leukocytes. To establish these CCN proteins as novel ligands of αMβ2, we demonstrated direct binding of the I domain of the integrin αM subunit (αMI) to immobilized CCN1 and CCN2 (12Schober 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 (213) Google Scholar). It is noteworthy that monocyte adhesion and αMI domain binding to CCN1 are blocked by anti-αM monoclonal antibodies as well as by soluble heparin. These findings suggest that the αMβ2 binding site may lie in close proximity to the heparin binding motifs within the C-terminal domain of CCN1. To gain further insight into the interaction of αMβ2 with CCN proteins, we sought to identify the αMβ2 binding site in CCN1. In the present study, we demonstrated αMβ2-dependent adhesion of monocytes to SSVKKYRPKYCGS present in the C-terminal domain of CCN1. Furthermore, the αMI domain binds specifically to this 13-residue sequence of CCN1 with a similar affinity as to the P2 (YSMKKTTMKIIPFNRLTIG) sequence, an αMβ2 binding site in the fibrinogen γ chain (25Ugarova T.P. Solovjov D.A. Zhang L. Loukinov D.I. Yee V.C. Medved L.V. Plow E.F. J. Biol. Chem. 1998; 273: 22519-22527Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). Our newly identified αMβ2 binding sequence in CCN1 bears no sequence homology to any known αMβ2 binding motif reported to date and may provide a target for blocking CCN1-αMβ2 interaction. Antibodies, Peptides, and Reagents—The anti-αM monoclonal antibody 2LPM19c was purchased from Dako Corp. MOPC 21, an isotype-matched control mouse IgG, was obtained from Sigma. For solid phase binding assays, anti-GST goat polyclonal antibody was from Amersham Biosciences, and horseradish peroxidase (HRP)-conjugated rabbit anti-goat IgG was from Sigma. For ELISA, HRP-conjugated donkey anti-rabbit antibody was from Pierce. An anti-peptide antibody (anti-CCN1367–381) against a peptide sequence corresponding to the extreme C terminus of CCN1 (F367PFYRLFNDIHKFRD381) was raised in rabbits and affinity-purified as previously described (13Grzeszkiewicz T.M. Lindner V. Chen N. Lam S.C. Lau L.F. Endocrinology. 2002; 143: 1441-1450Crossref PubMed Scopus (133) Google Scholar). Anti-domain I and anti-domain II polyclonal antibodies, raised in rabbits using GST fusion proteins linked to domain I or domain II of CCN1 as immunogens, were pre-absorbed with GST and then affinity-purified. In ELISA, both anti-domain I and anti-domain II antibodies reacted with full-length CCN1 as expected. Furthermore, these antibodies reacted specifically with their cognate CCN1 domains, and no cross-reactivity was observed (Table I).Table ISpecificity of anti-domain I and anti-domain II antibodies in ELISAAntibodiesA 490aMicrotiter wells were coated with 10 μg/ml full-length CCN1 and isolated domain I or domain II of CCN1 and then blocked with 1% BSA. Normal rabbit IgG, anti-domain I, or anti-domain II (100 nM each) was added and incubated for 1 h at 22 °C. After washing, bound antibodies were detected with an HRP-conjugated anti-rabbit IgG in an ELISA as described under “Materials and Methods.” Data shown are the means ± S.E. of triplicate determinations in one experiment.Full-length CCN1Domain IDomain IIIgG0.106 ± 0.0010.115 ± 0.0030.100 ± 0.001Anti-domain I2.203 ± 0.0011.999 ± 0.0130.131 ± 0.001Anti-domain II1.125 ± 0.0010.211 ± 0.0051.030 ± 0.005a Microtiter wells were coated with 10 μg/ml full-length CCN1 and isolated domain I or domain II of CCN1 and then blocked with 1% BSA. Normal rabbit IgG, anti-domain I, or anti-domain II (100 nM each) was added and incubated for 1 h at 22 °C. After washing, bound antibodies were detected with an HRP-conjugated anti-rabbit IgG in an ELISA as described under “Materials and Methods.” Data shown are the means ± S.E. of triplicate determinations in one experiment. Open table in a new tab Peptides sequences were represented by the single letter amino acid code (26IUPAC-IUBJ. Biol. Chem. 1968; 243: 3557-3559Abstract Full Text PDF PubMed Google Scholar). CCN1-H1 (YSSLKKGKKCSKTKKSPE), CCN1-H2 (SSVKKYRPKYCGS), CCN1-scrH2 (YRSCYSKVKPSGK), CCN2-H2 (TSVKTYRAKFCGV), and the fibrinogen P2 peptide (YSMKKTTMKIIPFNRLTIG) were synthesized by ResGen, Inc. For the production of anti-CCN1-H2 antibodies, rabbits were immunized with CCN1-H2 coupled to keyhole limpet hemocyanin (KLH) using the Imject® maleimide-activated mcKLH kit (Pierce). Anti-CCN1-H2 antibodies were affinity-purified on protein A-Sepharose and then on CCN1-H2-coupled Sepharose. Preimmune rabbit IgG was purified on protein A-Sepharose. Streptavidin (Sigma) was labeled with carrier-free Na125I (ICN Biomedicals, Inc.) using the Iodobeads iodination reagent (Pierce) to a specific activity of ∼0.3 μCi/μg. Protein Purification—Recombinant mouse CCN1, synthesized in a baculovirus expression system using Sf9 insect cells, was purified from serum-free conditioned media by Sepharose S chromatography as previously described (27Kireeva M.L. MO F.E. Yang G.P. Lau L.F. Mol. Cell. Biol. 1996; 16: 1326-1334Crossref PubMed Scopus (303) Google Scholar). The CCN1 truncation mutant lacking the C-terminal domain (CCN1ΔCT) was produced as a hexahistidine-tagged fusion protein and purified by nickel-agarose chromatography as described (10Grzeszkiewicz T.M. Kirschling D.J. Chen N. Lau L.F. J. Biol. Chem. 2001; 276: 21943-21950Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). Purified CCN1 and CCN1ΔCT were analyzed by SDS-polyacrylamide gel electrophoresis followed by Coomassie Blue staining and immunoblotting. Recombinant I domain of the integrin αM subunit was expressed as a fusion protein with GST (GST-αMI) and purified as described previously (28Yakubenko V.P. Solovjov D.A. Zhang L. Yee V.C. Plow E.F. Ugarova T.P. J. Biol. Chem. 2001; 276: 13995-14003Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Briefly, the coding region for human αMI domain sequence Asp132–Ala318 was amplified and inserted into the expression vector pGEX-4T-1 (Amersham Biosciences). Escherichia coli cells were transformed with the above recombinant vector, and protein expression was induced with 1mm isopropyl-β-d-thiogalactopyranoside for4hat37 °C. The GST-αMI fusion protein was affinity-purified from cell lysates using glutathione-agarose (Sigma). Isolation of Peripheral Blood Monocytes—Peripheral blood monocytes were isolated from human blood as previously described (12Schober 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 (213) Google Scholar). Acid-citrate-dextrose anticoagulated blood was collected from healthy donors and centrifuged to remove platelet-rich plasma. For isolation of mononuclear cells, the remaining packed red blood cells and buffy coat was diluted with phosphate-buffered saline (10 mm sodium phosphate, pH 7.35, 0.15 m NaCl) and centrifuged through a layer of Ficoll-Paque (Amersham Biosciences) at 400 × g for 60 min at 4 °C. The mononuclear cell layer was diluted in an equal volume of phosphate-buffered saline containing 2 mm EDTA, sedimented, and washed twice with modified Tyrode's buffer (10 mm Hepes, pH 7.35, 135 mm NaCl, 2.9 mm KCl, 12 mm NaHCO2,1mm MgCl2,1mm CaCl2, 0.1% dextrose, and 0.2% bovine serum albumin (BSA)) by centrifugation at 130 × g for 10 min. To further separate monocytes from lymphocytes, the mononuclear cells were suspended in modified Tyrode's buffer and subjected to centrifugation through a discontinuous density gradient of Percoll (Amersham Biosciences). Monocytes were isolated between a Percoll density of 1.047 and 1.050 g/ml, washed with modified Tyrode's buffer, and resuspended to a final concentration of 2–3 × 106 cells/ml. The purity of the monocyte preparation was greater than 80%, as measured by anti-CD14 staining in flow cytometry, and cell viability was more than 95% as judged by trypan blue exclusion. Cell Adhesion Assay—Microtiter wells (Immulon 2 Removawell strips, Dynex Technologies, Inc.) were coated with 10 μg/ml CCN1 or CCN1ΔCT protein or with 4 mg/ml peptide for 20 h at 4 °C. After protein or peptide coating, the wells were blocked with 0.2% polyvinyl alcohol (PVA) for 30 min at 37 °C. The amounts of immobilized CCN1 and CCN1ΔCT protein were quantified by an ELISA using anti-domain I and anti-CCN1367–381 antibodies as indicated. The amounts of immobilized peptides were measured by incubation for 2 h at 22 °C with 10 μm PEO-maleimide-activated biotin (Pierce), which reacts with the free sulfhydryl group in the cysteine residue of the peptides. After washing, the amounts of coupled biotin were quantified by incubation with 0.5 μm125I-labeled streptavidin, and bound radioactivity was measured by γ-counting. In cell adhesion experiments, isolated monocytes were activated with 20 μm ADP, added to the wells (100 μl/well), and incubated for 20 min at 37 °C. After washing to remove non-adherent cells, adherent cells were quantified using the acid phosphatase assay by incubation with the substrate solution (0.1 m sodium acetate, pH 5.5, 10 mm p-nitrophenylphosphate, and 0.1% Triton X-100; 100 μl/well) for 2 h at 37 °C (29Connolly D.T. Knight M.B. Harakas N.K. Wittwer A.J. Feder J. Anal. Biochem. 1986; 152: 136-140Crossref PubMed Scopus (182) Google Scholar). The reaction was stopped by the addition of 15 μlof1 n NaOH/well, and A 450 was measured. In inhibition studies, monocytes were preincubated with antibodies for 30 min at 37 °C before addition to microtiter wells. Binding of GST-αM I to Immobilized CCN1 and CCN-derived Peptides—Microtiter wells were coated with 30 μg/ml wild type or truncated CCN1 or 4 mg/ml synthetic peptides for 20 h at 4 °C and blocked with 0.2% PVA for 30 min at 37 °C. GST-αMI fusion protein was added to the wells and incubated for 1–2 h at 22 °C. Unbound GST-αMI was removed by washing with 30 mm Tris-HCl, pH 7.4, 0.2 m NaCl, 1 mm MgCl2, and 0.02% PVA. Bound GST-αMI was detected by an ELISA using anti-GST followed by an HRP-conjugated secondary antibody. Bound antibodies were detected using o-phenylenediamine dihydrochloride (Sigma) as the substrate. The reaction was stopped by the addition of 25 μl of 6 n HCl, and A 490 was measured. In inhibition experiments using an anti-αM monoclonal antibody, GST-αMI was preincubated with the antibody for 30 min at 37 °C before addition to the wells. In inhibition studies using anti-CCN1-H2 polyclonal antibodies, CCN1-coated wells were blocked with 1% BSA and preincubated with the antibodies for 30 min at 37 °C before the addition of the GST-αMI fusion protein. Binding proceeded for 30 min at 37 °C, and bound GST-αMI was detected as described above. In binding isotherm studies, input GST-αMI concentrations required for half-maximal binding of the fusion protein to immobilized peptides were estimated using the GraphPad Prism software in which the data was fitted to the one-site binding equation. The C-terminal Domain of CCN1 Is Required for Monocyte Adhesion and αM I Domain Binding—We previously identified αMβ2 as the major adhesion receptor on monocytes for CCN1 and CCN2 and showed that the I domain of the αM subunit bound specifically to both proteins (12Schober 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 (213) Google Scholar). Moreover, soluble heparin inhibited monocyte adhesion and αMI domain binding by interacting with the heparin binding motifs at the C-terminal domain of CCN1. The inhibitory effect of heparin suggests that the αMβ2 recognition site in CCN1 may be located in its C-terminal domain. To investigate this possibility, we performed monocyte adhesion and αMI domain binding experiments on a truncated CCN1 mutant that lacks the C-terminal domain (CCN1ΔCT) (10Grzeszkiewicz T.M. Kirschling D.J. Chen N. Lau L.F. J. Biol. Chem. 2001; 276: 21943-21950Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). Fig. 1A shows that monocytes adhered to wells coated with wild type CCN1 protein. By contrast, no cell adhesion to CCN1ΔCT-coated wells was observed. In agreement with the cell adhesion results, a GST fusion protein containing the αMI domain (GST-αMI) bound only to intact CCN1 but not to CCN1ΔCT (Fig. 1B). To determine whether similar amounts of CCN1 and CCN1ΔCT protein were immobilized onto microtiter wells, we performed an ELISA using two polyclonal antibodies raised against the entire domain I of CCN1 (anti-domain I) or a synthetic peptide corresponding to residue Phe367–Asp381 at the C terminus of CCN1 (anti-CCN1367–381). As shown in Fig. 1C, almost equal signals were obtained using the anti-domain I antibodies, indicating that similar amounts of CCN1 and CCN1ΔCT were immobilized. As expected, anti-CCN1367–381, directed against the extreme C terminus of CCN1, reacted with intact CCN1 but not with CCN1ΔCT, which lacks the C-terminal domain. Together, these results indicate that the C-terminal domain of CCN1 is required for both monocyte adhesion and αMI domain binding. Thus, the αMβ2 binding site likely resides within the C-terminal region of CCN proteins. The H2 Sequence in the CCN1 C-terminal Domain Supports Monocyte Adhesion—The inability of CCN1ΔCT to support monocyte adhesion and αMI domain binding suggests that the recognition site for αMβ2 is located in the C-terminal domain of CCN1. We focused on the first half of the C-terminal domain, because this region is highly homologous between CCN1 and CCN2, and both proteins support monocyte adhesion and αMI domain binding. Also, heparin has been shown to inhibit monocyte adhesion and αMI domain binding to CCN1 (12Schober 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 (213) Google Scholar), and therefore, we examined the possibility that αMβ2 binds directly to the heparin binding motifs in CCN1. In these studies we synthesized two peptides, CCN1-H1 and CCN1-H2, that correspond to the two heparin binding motifs of CCN1 (Fig. 2A). The peptides were immobilized onto microtiter wells, and their ability to support monocyte adhesion was examined. To determine the coating efficiency of CCN1-H1 and CCN1-H2 onto the wells, PEO-maleimide-activated biotin was allowed to react with the free sulfhydryl group in the single cysteine residue present in each peptide followed by detection with 125I-labeled streptavidin. Quantitation of bound radioactivity indicates that similar amounts of both peptides were immobilized onto the wells (Fig. 2B). In cell adhesion studies we found that monocytes adhered to CCN1-H2, but not to CCN1-H1 (Fig. 2C). Furthermore, monocyte adhesion to CCN1-H2 was completely blocked by 2LPM19c, an anti-αM monoclonal antibody, whereas control mouse IgG had no effect. Microscopic examination of the monocytes adherent to CCN1 protein and CCN1-H2 peptide revealed that the cells were well spread on both substrates (data not shown). Together, these results indicate that the H2 sequence in the C-terminal domain of CCN1 specifically mediates monocyte adhesion in an αMβ2-dependent manner. We previously reported that monocyte adhesion to CCN1 is markedly enhanced after cellular activation with ADP and formylmethionylleucylphenylalanine (12Schober 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 (213) Google Scholar). Enhanced adhesion of activated monocytes to CCN1 is likely due to inside-out signaling, resulting in increased αMβ2 affinity for CCN1 and/or mobilization of internal αMβ2 to the cell surface. In addition to inside-out signaling, the activation states of integrins can be modulated by extracellular divalent cations inasmuch as Mn2+ has been shown to increase the apparent affinity/avidity of multiple integrins including αMβ2 (30Li R. Rieu P. Griffith D.L. Scott D. Arnaout M.A. J. Cell Biol. 1998; 143: 1523-1534Crossref PubMed Scopus (123) Google Scholar). We, therefore, examined the effect of extracellular Mn2+ on monocyte adhesion to intact CCN1 protein and to the CCN1-H2 peptide. As expected, the addition of 1 mm Mn2+ to the cell suspension resulted in enhanced monocyte adhesion to CCN1 (Fig. 3). Likewise, monocyte adhesion to the CCN1-H2 peptide was also significantly enhanced in the presence of Mn2+. In a specificity control, monocytes were allowed to adhere to wells coated with a scrambled CCN1-H2 sequence (CCN1-scrH2). To confirm that CCN1-scrH2 was adsorbed onto microtiter wells, we performed binding of PEO-maleimide-activated biotin and 125I-labeled streptavidin as described above. Even though a higher amount of CCN1-scrH2 (95.6 ± 6.1 fmol/well) was coated onto the wells as compared with CCN1-H2 (43.6 ± 0.9 fmol/well), no significant cell adhesion to CCN1-scrH2-coated wells was observed in the absence and presence of Mn2+. The ability of Mn2+ to enhance monocyte adhesion to CCN1 and CCN1-H2, but not to CCN1-scrH2, indicates that activated αMβ2 binds with a higher affinity to intact CCN1 and to the CCN1-H2 sequence. Binding of αM I Domain to the H2 Sequences in CCN proteins—The above cell adhesion data suggest that the CCN1-H2 sequence, but not CCN1-H1, serves as a binding site for integrin αMβ2. To corroborate cell adhesion studies with receptor binding, we performed αMI domain binding experiments on immobilized CCN1-H1 and CCN1-H2. Fig. 4A shows that GST-αMI bound directly to wells coated with the CCN1 protein or with the CCN1-H2 peptide, but not to wells coated with the CCN1-H1 peptide (filled bars). As controls, GST itself did not bind to CCN1, CCN1-H1, or CCN1-H2 (open bars). To demonstrate binding specificity, we tested the effect of 2LPM19c on αMI domain binding. As shown in Fig. 4B, inhibition of GST-αMI binding to CCN1-H2 was observed with 2LPM19c (anti-αM) but not with an isotype-matched mouse IgG. These results indicate that integrin αMβ2 binds directly to the CCN1-H2 sequence to mediate monocyte adhesion to CCN1 protein. In addition to CCN1, we previously reported that the αMI domain also binds to CCN2, another CCN family member highly homologous to CCN1 (12Schober 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 (213) Google Scholar). Therefore, we performed solid phase binding studies to examine whether GST-αMI also binds to the corresponding H2 sequence in CCN2. Fig. 5A shows the H2 sequences in CCN1 and CCN2 with the non-conserved residues underlined. The αMI domain was found to bind dose dependently to both CCN1-H2 and CCN2-H2 peptides, whereas no binding was observed with wells coated with the CCN1-scrH2 sequence (Fig. 5B). Using the GraphPad Prism software, we estimated that half-maximal binding of GST-αMI to immobilized CCN1-H2 and CCN2-H2 occurred at input GST-αMI concentrations of 47.2 ± 6.4 and 128.0 ± 26.3 nm (means ± S.E.; n = 5 for CCN1-H2 and n = 3 for CCN2-H2), respectively. Thus, the results of the binding isotherms indicate that the αMI domain binds with a higher affinity to CCN1-H2 than to CCN2-H2 (Student's t test, p < 0.01). Consistently, we previously showed that monocytic THP-1 cells adhere more strongly to CCN1 than to CCN2 (12Schober 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 (213) Google Scholar). It has been reported th" @default.
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• W2087823284 title "Identification of a Novel Integrin αMβ2 Binding Site in CCN1 (CYR61), a Matricellular Protein Expressed in Healing Wounds and Atherosclerotic Lesions" @default.
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