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• W2040536490 abstract "Type IV collagen (COL-IV) interacts with a variety of cell types. We present evidence that human mesangial cells (HMC) bind directly to COL-IV, its major triple helical domain, and the main non-collagenous, NC1 domain. A synthetic peptide, HEP-III, and its triple helical counterpart (THP-III), previously reported to be a heparin-binding domain, also promoted ≈15% adhesion of HMC. HMC bound to solid-phase-immobilized, intact COL-IV (≈75%), isolated NC1 domain (≈15%), and a pepsin-derived triple helical fragment,which lacks Hep-III (≈65%). We further examined inhibition of HMC adhesion to COL-IV and its domains by using anti-integrin antibodies. Blocking monoclonal antibodies against the α2 integrin resulted in 70% inhibition of adhesion to COL-IV and 80% inhibition to HEP-III. Moderate inhibition was observed on the NC1 and triple helical fragments. Anti-α1 antibodies inhibited the binding of HMC to COL-IV, the NC1, and triple helical domains, but not to peptide HEP-III. Anti-β1 antibodies inhibited almost completely (>95%) the adhesion to COL-IV, the NC1, and triple helical fragments; inhibition on HEP-III was ≈30%. Affinity chromatography studies with solid-phase HEP-III and mesangial cell lysate also demonstrated the presence of integrin α2β1along with α3β1. We conclude that α2β1 and α1β1integrins mediate HMC adhesion to COL-IV. Peptide HEP-III is a major, specific site for α2 integrin-mediated binding of mesangial cells to COL-IV. Both the α1β1and α2β1 integrins interact with the NC1 and triple helical fragments of COL-IV. Therefore, we demonstrate that several sites for integrin-mediated interactions exist on several collagenous and non-collagenous domains of COL-IV. Type IV collagen (COL-IV) interacts with a variety of cell types. We present evidence that human mesangial cells (HMC) bind directly to COL-IV, its major triple helical domain, and the main non-collagenous, NC1 domain. A synthetic peptide, HEP-III, and its triple helical counterpart (THP-III), previously reported to be a heparin-binding domain, also promoted ≈15% adhesion of HMC. HMC bound to solid-phase-immobilized, intact COL-IV (≈75%), isolated NC1 domain (≈15%), and a pepsin-derived triple helical fragment,which lacks Hep-III (≈65%). We further examined inhibition of HMC adhesion to COL-IV and its domains by using anti-integrin antibodies. Blocking monoclonal antibodies against the α2 integrin resulted in 70% inhibition of adhesion to COL-IV and 80% inhibition to HEP-III. Moderate inhibition was observed on the NC1 and triple helical fragments. Anti-α1 antibodies inhibited the binding of HMC to COL-IV, the NC1, and triple helical domains, but not to peptide HEP-III. Anti-β1 antibodies inhibited almost completely (>95%) the adhesion to COL-IV, the NC1, and triple helical fragments; inhibition on HEP-III was ≈30%. Affinity chromatography studies with solid-phase HEP-III and mesangial cell lysate also demonstrated the presence of integrin α2β1along with α3β1. We conclude that α2β1 and α1β1integrins mediate HMC adhesion to COL-IV. Peptide HEP-III is a major, specific site for α2 integrin-mediated binding of mesangial cells to COL-IV. Both the α1β1and α2β1 integrins interact with the NC1 and triple helical fragments of COL-IV. Therefore, we demonstrate that several sites for integrin-mediated interactions exist on several collagenous and non-collagenous domains of COL-IV. COL-IV, 1The abbreviations used are: COL-IV, type IV collagen; HMC, human mesangial cells; pepsin-IV, pepsin-derived triple helical fragments of COL-IV; BSA, bovine serum albumin; mAb, monoclonal antibody; EHS, Engelbreth-Holm-Swarm; DMEM, Dulbecco's modified Eagle's medium.1The abbreviations used are: COL-IV, type IV collagen; HMC, human mesangial cells; pepsin-IV, pepsin-derived triple helical fragments of COL-IV; BSA, bovine serum albumin; mAb, monoclonal antibody; EHS, Engelbreth-Holm-Swarm; DMEM, Dulbecco's modified Eagle's medium. originally isolated in an intact form from the Engelbreth-Holm-Swarm (EHS) tumor (1Kleinman H.K. McGarvey M.L. Liotta L.A. Robey P.G. Tryggvason K. Martin G.R. Biochemistry. 1982; 21: 6188-6193Crossref PubMed Scopus (953) Google Scholar), is a large glycoprotein (M r 500,000), which has the ability to polymerize into a network (2Timpl R. Wiedemann H. van Delden V. Furthmayr H. Kuhn K. Eur. J. Biochem. 1981; 120: 203-211Crossref PubMed Scopus (643) Google Scholar) on which other components such as laminin, entactin/nidogen, and heparan sulfate proteoglycan can bind and assemble (3Yurchenco P.D. Schittny J.C. FASEB J. 1990; 4: 1577-1590Crossref PubMed Scopus (782) Google Scholar). COL-IV also participates in the interaction of basement membranes with cells (4Aumailley M. Timpl R. J. Cell Biol. 1986; 103: 1569-1575Crossref PubMed Scopus (142) Google Scholar). This glycoprotein is composed of three chains ((α1)2α2), but several additional isoform chains also exist), each consisting of a pepsin-resistant, discontinuous triple helical domain and a major non-collagenous NC1 domain at the carboxyl-terminal end. COL-IV contains interruptions in Gly-X-Y repeats of the collagenous domain; 21 of these occur in α1(IV) and 23 in the α2(IV) chain. One interruption located at 100 nm from the amino end of the α1(IV) has been made synthetically (peptide Hep-III) and was described by our group to mediate heparin and cell binding (5Koliakos G.G. Kouzi-Koliakos K. Furcht L.T. Reger L.A. Tsilibary E.C. J. Biol. Chem. 1989; 264): 2313-2323Abstract Full Text PDF PubMed Google Scholar, 6Koliakos G.G. Skubitz A.P.N. Pirner M.A. Tsilibary E.C. J. Cell Biol. 1989; 109 (abstr.): 200Google Scholar). The sequence of this peptide (GEFYFDLRLKGDK) represents a pepsin-sensitive site (7Schuppan D. Timpl R. Glanville R.W. FEBS Lett. 1980; 115: 297-300Crossref PubMed Scopus (64) Google Scholar, 8Glanville R.W. Rauter A. Hoppe-Seyler's Z. Physiol. Chem. 1981; 362: 943-951Crossref PubMed Scopus (25) Google Scholar). Collagens have been reported to interact with various cells at multiple interaction sites along the triple helix, in native or denatured form (9Klein G. Langegger M. Timpl R. Ekblom P. Cell. 1988; 55: 331-341Abstract Full Text PDF PubMed Scopus (384) Google Scholar, 10Montesano R. Orci L. Vassali P. J. Cell Biol. 1983; 97: 1648-1652Crossref PubMed Scopus (497) Google Scholar, 11Rubin K. Hook M. Obrink B. Timpl R. Cell. 1981; 24: 463-470Abstract Full Text PDF PubMed Scopus (134) Google Scholar). The major collagen receptors have been demonstrated to belong to the β1 subgroup of the integrin family (12Wayner E.A. Carter W.G. J. Cell Biol. 1987; 105: 1873-1884Crossref PubMed Scopus (537) Google Scholar, 13Santoro S.A. Rajpura S.M. Staatz W.D. Woods Jr., V.L. Biochem. Biophys. Res. Commun. 1988; 153: 217-223Crossref PubMed Scopus (87) Google Scholar), namely α1β1, α2β1, and α3β1. In COL-IV, using isolated integrins and fragments of COL-IV, α1β1 and α2β1integrin binding sites have been mapped to the CnBr fragment CB3[IV] (a 150-amino acid segment of COL-IV that is located 100 nm from the amino-terminal end) (14Vandenberg P. Kern A. Ries A. Luckenbill L. Mann K. Kuhn K. J. Cell Biol. 1991; 113: 1475-1483Crossref PubMed Scopus (195) Google Scholar). Further digestion of the CB3[IV] fragment by trypsin yields shorter triple helical fragments. Kern and co-workers (15Kern A. Eble J., R. Goblik J. Kuhn K. Eur. J. Biochem. 1993; 215: 151-159Crossref PubMed Scopus (178) Google Scholar), using solid-phase and inhibition assays demonstrated that α1β1 and α2β1use two distinct binding sites located on two neighboring segments of COL-IV. The α2β1 receptor was observed to have more than one site of binding, one of which includes an interrupt in the triple helix located about 100 nm from the amino terminus of the molecule. Also, an α2 integrin binding site has been localized to the CB3 fragment of the α1(I) collagen chain (16Staatz W.D. Walsh J.J. Pexton T. Santoro S.A. J. Biol. Chem. 1990; 265: 4778-4781Abstract Full Text PDF PubMed Google Scholar). In this report, we have examined integrin-mediated interactions between human mesangial cells (HMC) and COL-IV, because COL-IV is the predominant molecule in the matrix surrounding these cells (17Kim Y. Kleppel M.M. Butkowski R. Mauer S.M. Weislander J. Michael A.F. Am. J. Pathol. 1991; 138: 413-420PubMed Google Scholar). We have used primary cell cultures and EHS-derived COL-IV. EHS-derived COL-IV has the same chain composition as the COL-IV surrounding mesangial cells in the glomerulus (17Kim Y. Kleppel M.M. Butkowski R. Mauer S.M. Weislander J. Michael A.F. Am. J. Pathol. 1991; 138: 413-420PubMed Google Scholar). In addition, various studies have localized the β1 family of integrins to the kidney (18Hemler M.E. Immunol. Today. 1988; 9: 109-113Abstract Full Text PDF PubMed Scopus (258) Google Scholar) and to mesangial cells (19Cosio F.G. Am. J. Kidney Dis. 1992; 20: 294-305Abstract Full Text PDF PubMed Scopus (37) Google Scholar). The cell surface receptors α1β1 and α2β1specifically mediate adhesion of cells to COL-IV (14Vandenberg P. Kern A. Ries A. Luckenbill L. Mann K. Kuhn K. J. Cell Biol. 1991; 113: 1475-1483Crossref PubMed Scopus (195) Google Scholar). Monoclonal antibodies against integrins that perturb ligand-mediated cell adhesion have been used to identify integrin molecules responsible for various interactions with matrix molecules or other cells. We have used the former approach to map the sites of interaction of human mesangial cells to COL-IV. The β1 family of integrins was originally termed VLA or very late antigens and consists of at least six different members (18Hemler M.E. Immunol. Today. 1988; 9: 109-113Abstract Full Text PDF PubMed Scopus (258) Google Scholar). The β1 integrins are important mediators of cell adhesion and migration; these receptors also influence cell proliferation, differentiation, and developmental processes (20Hynes R. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (8941) Google Scholar, 21Ruoslahti E. J. Clin. Invest. 1991; 87: 1-5Crossref PubMed Scopus (1477) Google Scholar, 22Menko A.S. Boettiger D. Cell. 1987; 51: 51-57Abstract Full Text PDF PubMed Scopus (308) Google Scholar, 23MacKrell A.J. Blumberg B. Haynes S.R. Fessler J.H. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2633-2637Crossref PubMed Scopus (164) Google Scholar). Additionally, integrins have a central role in extracellular matrix assembly (23MacKrell A.J. Blumberg B. Haynes S.R. Fessler J.H. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2633-2637Crossref PubMed Scopus (164) Google Scholar, 24McDonald J.A. Annu. Rev. Cell Biol. 1988; 4: 183-207Crossref PubMed Scopus (219) Google Scholar). Cells may bind more than one matrix molecule and vice versa. Many cells utilize different integrins to attach to different macromolecules; often integrins are induced on the cell surface after exposure of a cell to a matrix molecule (25Roman J. LaChance R.M. Broekelmann T.J. Kennedy C.J.R. Wayner E.A. Carter W.G. McDonald J.A. J. Cell Biol. 1989; 108: 2529-2543Crossref PubMed Scopus (99) Google Scholar). Also, the same cell may simultaneously use different integrins to interact with the same matrix component (26von der Mark K. von der Mark H. Goodman S. Kidney Int. 1992; 41: 632-640Abstract Full Text PDF PubMed Scopus (45) Google Scholar). This redundancy is important for understanding the mechanism of ligand binding as well as the functional and signaling specificity of the various receptors (27Humphries M.J. Mould A.P. Tuckwell D.S. Bioassays. 1993; 15: 391-397Crossref PubMed Scopus (54) Google Scholar). We report here that a sequence contained in a synthetic peptide, HEP-III, positioned ≈100 nm from the amino-terminal end of the α1(IV) contains a major α2β1integrin binding site of COL-IV. Monoclonal antibodies against the α2 and β1 chains of the integrin family of receptors significantly inhibit mesangial cell attachment to this site in COL-IV, indicating that integrin α2β1 of HMC binds specifically to this sequence in COL-IV. Other well characterized COL-IV fragments, including the triple helix and the NC1 domain, were observed to sustain cell adhesion via a combination of α1β1 and α2β1integrins. HMC were isolated from the kidneys of 19–22-week-old fetuses and characterized as described previously (28Striker G.E. Striker L. J. Lab. Invest. 1985; 53: 122-131PubMed Google Scholar). The cells were cultured in Dulbecco's minimal essential medium (DMEM) supplemented with 20% fetal calf serum (Sigma), 15 mmHEPES, 25 mm glucose, and antibiotics amphotericin (0.25 μg/ml), penicillin G (100 IU/ml), and streptomycin (100 μg/ml). Cells were used through passage 5–9, grown in T-75 flasks till 75–80% confluence, and then metabolically labeled for 18 h with 0.5 mCi of [35S]methionine per T-75 flask. [35S]Methionine was obtained from NEN Life Science Products Inc. COL-IV was isolated from (EHS) as described previously (29Tsilibary E.C. Charonis A.S. J. Cell Biol. 1986; 103: 2467-2473Crossref PubMed Scopus (66) Google Scholar). The major triple helix-rich domain, which lacks the NC1 and 7 S domains, was obtained by a light digestion of EHS-derived COL-IV with pepsin (Cooper Biomedical Inc.) as described by Yurchenco and Furthmayr (30Yurchenco P.D. Furthmayr H. Biochemistry. 1984; 23: 1839-1850Crossref PubMed Scopus (244) Google Scholar). The size of this fragment is ≈400 nm (30Yurchenco P.D. Furthmayr H. Biochemistry. 1984; 23: 1839-1850Crossref PubMed Scopus (244) Google Scholar). This fragment also lacks some pepsin-sensitive interruptions in the triple helical domain, including the sequence contained in peptide Hep-III (31Timpl R. Bruckner P. Fietzek P. Eur. J. Biochem. 1979; 95: 255-263Crossref PubMed Scopus (94) Google Scholar). The major non-collagenous domain or NC1 of COL-IV was obtained by digestion of EHS-derived COL-IV with collagenase (CLSPA Cooper Biomedical Inc.) as described previously (29Tsilibary E.C. Charonis A.S. J. Cell Biol. 1986; 103: 2467-2473Crossref PubMed Scopus (66) Google Scholar, 32Tsilibary E.C. Charonis A.S. Reger L.A. Wohlhueter L.A. Furcht L.T. J. Biol. Chem. 1988; 263: 4302-4308Abstract Full Text PDF PubMed Google Scholar). Peptide HEP-III was synthesized by the method of Barany and Merrifield (33Barany G. Merrifield R.B. Gross E. Meienhofer J. The Peptides. Academic Press, Orlando, FL1980: 1-284Google Scholar) on a solid-phase support as described previously (5Koliakos G.G. Kouzi-Koliakos K. Furcht L.T. Reger L.A. Tsilibary E.C. J. Biol. Chem. 1989; 264): 2313-2323Abstract Full Text PDF PubMed Google Scholar). Also, a solid-phase methodology was used for the synthesis of the triple helical polypeptide THP-III incorporating native sequences from the α1(IV) chain (34Fields C.G. Lovdahl C.M. Miles A.J. Matthias Hagen V.L. Fields G.B. Biopolymers. 1993; 33: 1685-1707Crossref Scopus (91) Google Scholar). Anti-NC1 antibodies were produced as described previously (29Tsilibary E.C. Charonis A.S. J. Cell Biol. 1986; 103: 2467-2473Crossref PubMed Scopus (66) Google Scholar). Briefly, isolated NC1 in complete Freund's adjuvant was injected subcutaneously in female New Zealand rabbits three times at two-week intervals. Two weeks after the last injection the animals were bled, and the antiserum was found to be reactive with the NC1 domain. Monovalent Fab fragments were prepared by papain digestion of the IgG fraction of the antiserum. 96-well Immulon 1 plates (Fisher Scientific Co.) were coated with 50 μl of COL-IV, pepsin-derived triple helical fragment, and NC1 domain in serial dilutions starting from 25, 25, and 40 μg/ml overnight, respectively. The triple helical peptide (THP-III) and HEP-III peptides were coated in 50 μl of phosphate-buffered saline in serial dilutions starting from 200 μg/ml. We have previously determined that under these conditions, ≈45–50% of each of these fragments adhere to plastic (5Koliakos G.G. Kouzi-Koliakos K. Furcht L.T. Reger L.A. Tsilibary E.C. J. Biol. Chem. 1989; 264): 2313-2323Abstract Full Text PDF PubMed Google Scholar, 35Herbst T.J. McCarthy J.B. Tsilibary E.C. Furcht L.T. J. Cell Biol. 1988; 106: 1365-1373Crossref PubMed Scopus (176) Google Scholar). In order to block the remaining reactive sites the plates were treated with 200 μl of BSA at 2 mg/ml for 2 h at 37 °C. The [35S]methionine-labeled cells were detached from culture flasks by incubation with 0.05% trypsin and 0.02% EDTA for 2 min at 37 °C, washed twice with DMEM, and resuspended to the appropriate concentration in binding buffer (DMEM, 125 mm HEPES, 2 mg/ml BSA at pH 7.4). A 50-μl suspension containing 5000 cells was added to each well. The plates were incubated at 37 °C in a humidified incubator for approximately 30 min. The cells were then washed three times with binding buffer, and 100 μl of “lysis” buffer (0.5 m NaOH, 1% SDS in distilled water) was added to each well. The plates were then incubated at 60 °C for 30 min and 45 min in the case of the NC1 fragment. The lysate was transferred to scintillation vials and counted. The binding of HMC to albumin (control, 1–2%) was subtracted. The data were plotted after determining the equimolar amounts of COL-IV and fragments to compare the ability of each component to bind mesangial cells, expressed as a mean of quadruplicate wells ± SD of the percentage of the total input counts/min. Competition of mesangial cell adhesion to solid-phase COL-IV (2.5 mg/ml) was performed with peptides HEP-III and THP-III. Serial dilutions of peptides were preincubated with HMC for 30 min, and then the cells with peptide were added to 96-well plates coated with COL-IV and incubated for a further 30 min. The non-adherent cells were washed off, and adherent cells were processed as described earlier. The data were means of quadruplicate wells and were expressed as a percentage of cell adhesion in the absence of peptide. Mouse mAbs to the integrin receptors α1 (SR84), α2 (P1H5), α3 (P3D11), α4 (P4G9), α5(P3D10), and β1 (P5D2) have been previously described as adhesion-inhibiting antibodies in various systems (36Wayner E.A. Gil S.G. Murphy F. Wilke M.S. Carter W.G. J. Cell Biol. 1993; 121: 1141-1152Crossref PubMed Scopus (152) Google Scholar, 37Rettig W.J. Dracopoli N.C. Goetzger T.A. Spengler B.A. Biedler J.L. Oettgen H.F. Old H.F. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 6437-6441Crossref PubMed Scopus (74) Google Scholar). A mAb directed to a cell surface HLA determinant was used as a negative control (W6/32, HB95; American Type Culture Collection, Rockville, MD). W6/32 bound to the surface of cultured mesangial cells but did not influence adhesion to COL-IV. Hybridoma culture supernatant or ascites fluid used in inhibition experiments was quantitated for antibody content by the technique of quantitative enzyme-linked immunosorbent assay. Integrins α1, α2, α3, α5, and β1 have been previously reported to be present on mesangial cells (as determined by flow cytometry (38Setty S. Anderson S.S. Wayner E.A. Kim Y. Clegg D.O. Tsilibary E.C. Cell Adhesion Commun. 1995; 3: 187-200Crossref PubMed Scopus (21) Google Scholar)). Subunits α4 and α6were not expressed on these cells. The role of these integrins in adhesion of mesangial cells to COL-IV was examined with an inhibition assay using monoclonal antibodies against several integrin subunits. These were added to solid-phase COL-IV along with [35S]methionine-labeled mesangial cells. The mixtures were incubated for 30 min at 37 °C. W6/32 supernatant or ascites was used as a control. Inhibition of adhesion, using saturation amounts of antibodies, was seen only with α1, α2, and β1 integrin subunits. Therefore, the inhibition pattern of COL-IV and its fragments was further studied using these antibodies. All inhibition assays in this study were done in quadruplicate, and the standard error was in all instances <5%. To confirm the role of the NC1 domain in cell adhesion, the adhesion of HMC to COL-IV was studied after blocking the NC1 domain with anti-NC1 antibody. Solid-phase COL-IV, at a concentration of 2.5 μg/ml, was preincubated with anti-NC1 antibody at a concentration of 1.25 μg/ml. Radiolabeled HMC were added for 30 min. Non-adherent cells were washed off, and adherent cells were quantitated as described previously. Adhesion of HMC in the presence of anti-NC1 antibody was compared with adhesion to COL-IV in the absence of antibody. 96-well plates were coated overnight with 50 μl of COL-IV and a pepsin-derived triple helical fragment at 2.5 μg/ml, NC1 at 10 μg/ml, and HEP-III and THP-III at 50 μg/ml. These coating concentrations were selected, based on direct cell adhesion assays as described above, using the middle of the log phase. The plates were incubated with 2% BSA in phosphate-buffered saline to block the remaining reactive sites on plastic for 2 h, and then serial dilutions of hybridoma culture supernatant containing known quantities of antibody were added to each well in quadruplicate. W6/32 supernatant or ascites was used as a control. Inhibition of adhesion on COL-IV was seen only with α1, α2, and β1anti-integrin antibodies, so the inhibition pattern of COL-IV fragments was studied using these antibodies. We used an enzyme-linked immunosorbent assay approach to determine the quantity of antibody in the hybridoma culture supernatant (39Coligan J.E. et al.Current Protocols in Immunology. 1992; (Section 2.1, Suppl. 4, CPI)Google Scholar). The quantity of antibody required to saturate the binding sites on human mesangial cells was determined by flow cytometry. The starting concentration of all antibodies used in the inhibition assays was consistently well above the saturating concentration, as determined by flow cytometry.35S-Labeled cells were processed as described above and were added to each well in 50 μl (5000/well). The adhesion assay was allowed to proceed for 30 min. Then, non-adherent cells were washed off and bound cells were quantitated as described previously. Data were expressed as a percentage of maximal binding observed in the absence of antibody. HEP-III peptide was coupled to activated CH-Sepharose (Amersham Pharmacia Biotech) (40Miles A.J. Knutson J.R. Skubitz A.P.N. Furcht L.T. McCarthy J.B. Fields G.B. J. Biol. Chem. 1995; 270: 29047-29050Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). 30 mg of high pressure liquid chromatography-purified peptide was dissolved in 200 μl of Me2SO and diluted to 5 ml with 15 mm sodium carbonate and 35 mm sodium bicarbonate (pH 8.6). The peptide solution was added to 3 ml of preswollen beads and mixed overnight at 4 °C. A mock column was made in parallel without peptide, for use as a control. Unbound peptide was removed with previously used diluent, and reactive sites were hydrolyzed at pH 8.0 with 0.1 m Tris-HCl for 2 h. HMC were metabolically labeled with [35S]methionine as described earlier and lysed with buffer (50 mm Tris-HCl, pH 7.4, 50 mmoctyl-β-d-glucopyranoside, 15 mm NaCl, 1 mm MgCl2, 1 mm MnCl2, 1 mm CaCl2, 1 mm N-ethylmaleimide, and 1 mmphenylmethylsulfonyl fluoride for 30 min at 4 °C. Following ultracentrifugation at 12,000 rpm for 30 min, the cell lysate was precleared with the mock-coupled Sepharose beads as a slurry by mixing overnight at 4 °C. The lysate was then incubated with the peptide-Sepharose beads overnight at 4 °C. The peptide-Sepharose beads were then packed into a column, and the column was washed with lysis buffer and eluted with 20 mm EDTA in lysis buffer lacking the cations. Eluates were incubated with anti-integrin antibodies to immunoprecipitate specific integrins (36Wayner E.A. Gil S.G. Murphy F. Wilke M.S. Carter W.G. J. Cell Biol. 1993; 121: 1141-1152Crossref PubMed Scopus (152) Google Scholar). Elution fractions were electrophoresed by 7.5% SDS-polyacrylamide gel electrophoresis and analyzed by autoradiography. Mesangial cell adhesion to the main fragments of COL-IV (the major triple helix and the NC1 domain) was compared with that of the intact molecule in a solid-phase assay. In dose-response cell binding experiments the highest percent adhesion was observed when COL-IV was plated at a concentration of 25 μg/ml and did not increase thereafter. Maximally, approximately 70–80% of the cells adhered on COL-IV (Table I). 2.5 μg/ml COL-IV promoted 30% adhesion of added mesangial cells (Fig.1 A). In similar experiments the pepsin-derived triple helical fragment bound a maximum of ≈65% cells, and NC1 bound ≈15% cells (Fig. 1 A); HEP-III and THP-III peptides bound ≈15% cells (Fig. 1 B). In the competition experiments we used 2.5 μg/ml COL-IV, 2.5 μg/ml pepsin-IV, 10 μg/ml NC1, and 50 μg HEP-III to promote slightly less than half-maximal cell binding. We also examined whether HEP-III in solution competed for the binding of mesangial cells to intact COL-IV. HMC were added in the presence of either peptides HEP-III or THP-III to solid-phase-adsorbed COL-IV (Fig. 1 C). Both forms of peptide inhibited cell adhesion in a concentration-dependent manner. THP-III inhibited HMC adhesion slightly better than HEP-III. In other experiments, when the main NC1 site in intact solid-phase COL-IV was blocked with anti-NC1 antibodies, there was a reduction in adhesion of HMC to COL-IV (≈15%) as compared with the absence of specific antibodies to NC1 (Fig. 2).Table IMesangial cell adhesion to COL-IV and fragmentsSubstrateMaximum % of cell adhesionInhibition of adhesionα1α2β1COL-IV75507095Pepsin-IV65802095NC115754595HEP-III15108030 Open table in a new tab Figure 2Inhibition of human mesangial cell adhesion to COL-IV in the presence of anti-NC1 and various anti-integrin antibodies. Antibody to HLA (W6/32) was used as a control. Only antibodies against the α1, α2 and β1 subunits competed for the binding of mesangial cells to COL-IV. Antibodies to the integrin subunits α3and α5, present on mesangial cells, did not inhibit binding. The binding of mesangial cells to COL-IV in the presence of an antibody to the NC1 domain was ≈15% less than in the presence of control.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The role of various integrins in mediating the adhesion of mesangial cells to COL-IV was examined with inhibition assays. In these experiments, monoclonal antibodies against several integrin subunits (reported previously to inhibit adhesion of different cells to various substrates (36Wayner E.A. Gil S.G. Murphy F. Wilke M.S. Carter W.G. J. Cell Biol. 1993; 121: 1141-1152Crossref PubMed Scopus (152) Google Scholar, 37Rettig W.J. Dracopoli N.C. Goetzger T.A. Spengler B.A. Biedler J.L. Oettgen H.F. Old H.F. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 6437-6441Crossref PubMed Scopus (74) Google Scholar)) were added to solid-phase COL-IV along with mesangial cells. The mixtures were incubated for 30 min at 37 °C. Percent adhesion in the presence of serial dilutions of these antibodies was then determined. From the examined panel of α1, α2, α3, α4, α5, and β1 anti-integrin antibodies, only antibodies against the α1, α2, and β1 competed for the binding of mesangial cells to COL-IV. The maximal observed inhibition in the presence of anti-β1 was ≈95%, in the presence of anti-α1 it was ≈50%, and in the presence of anti-α2 it was ≈70% (Fig. 2, Table I). Antibodies to α1 and α2 together caused inhibition to the same extent as β1. Antibodies to α4 did not result in inhibition, as expected, since α4 is not expressed by mesangial cells (38Setty S. Anderson S.S. Wayner E.A. Kim Y. Clegg D.O. Tsilibary E.C. Cell Adhesion Commun. 1995; 3: 187-200Crossref PubMed Scopus (21) Google Scholar). Based on these assays, the α1β1 and α2β1integrin receptors mediate the adhesion of human mesangial cells to COL-IV. We further determined whether there was preferential use of α1β1 or α2β1receptors by certain regions of the molecule. In order to obtain these data the inhibition assays were repeated with triple helical and NC1 fragments, as well as peptide HEP-III as substrates immobilized on a solid surface. Anti-β1 antibodies abolished adhesion to triple helical fragments and NC1 domains almost completely when used at the highest concentration. This is consistent with the previous observation that inhibition with β1 was almost complete on intact COL-IV. Only 30% inhibition was seen on peptide HEP-III (Fig. 3, A–C). Antibodies to the α2 integrin subunit inhibited adhesion to triple helical fragments only minimally (≈20%), even at the highest concentration, but effectively inhibited HMC adhesion to NC1 (≈60% at the highest concentration) and HEP-III (≈80% inhibition at the highest concentration) (Fig. 3, A–C). Anti-α1 integrin antibodies also inhibited adhesion to pepsin-derived triple helical fragments of COL-IV and NC1, but inhibition of adhesion to HEP-III was not significant, even at the highest concentration used. When anti-α1 and anti-α2 were combined, inhibition of adhesion to all fragments was almost complete (Fig. 3, A–C). The HEP-III peptide was immobilized to CH-Sepharose, and affinity chromatography was performed with a [35S]methionine-labeled extract of HMC. Equal counts of the eluate obtained with 20 mm EDTA were incubated with 10 μg/ml mAb against α1, α2, α3, β1 integrin subunits and normal mouse IgG as control. Immunoprecipitated proteins were analyzed by 7.5% SDS-polyacrylamide gel electrophoresis with detection by autoradiography. Immunoprecipitation with anti-β1integrin subunit mAb resulted in detection of 116- and 130-kDa proteins (Fig. 4). Similarly, immunoprecipitation of the same eluant with anti-α2 and α3integrin subunit mAb resulted in detection of 116- and 130-kDa proteins (Fig. 4). The molecular masses correspond to the α2, α3 (130 kDa), and β1 (116 kDa) integrin subunits (36Wayner E.A. Gil S.G. Murphy F. Wilke M.S. Carter W.G. J. Cell Biol. 1993" @default.
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• W2040536490 title "Interactions of Type IV Collagen and Its Domains with Human Mesangial Cells" @default.
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