Interactions of Type IV Collagen and Its Domains with Human Mesangial Cells*

Suman SettyDagger §, Youngki Kim, Gregg B. FieldsDagger , Dennis O. Cleggparallel , Elizabeth A. Wayner**, and Effie C. TsilibaryDagger Dagger Dagger

From the Departments of Dagger  Laboratory Medicine and Pathology and  Pediatrics, School of Medicine, University of Minnesota, Minneapolis, Minnesota 55455, Dagger Dagger  Institute of Biology, NRCPS Demokritos, Athens, Greece, ** Fred Hutchinson Cancer Center, Seattle, Washington, and parallel  Department of Biological Sciences, University of California, Santa Barbara, California

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 approx 15% adhesion of HMC. HMC bound to solid-phase-immobilized, intact COL-IV (approx 75%), isolated NC1 domain (approx 15%), and a pepsin-derived triple helical fragment,which lacks Hep-III (approx 65%). We further examined inhibition of HMC adhesion to COL-IV and its domains by using anti-integrin antibodies. Blocking monoclonal antibodies against the alpha 2 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-alpha 1 antibodies inhibited the binding of HMC to COL-IV, the NC1, and triple helical domains, but not to peptide HEP-III. Anti-beta 1 antibodies inhibited almost completely (>95%) the adhesion to COL-IV, the NC1, and triple helical fragments; inhibition on HEP-III was approx 30%. Affinity chromatography studies with solid-phase HEP-III and mesangial cell lysate also demonstrated the presence of integrin alpha 2beta 1 along with alpha 3beta 1. We conclude that alpha 2beta 1 and alpha 1beta 1 integrins mediate HMC adhesion to COL-IV. Peptide HEP-III is a major, specific site for alpha 2 integrin-mediated binding of mesangial cells to COL-IV. Both the alpha 1beta 1 and alpha 2beta 1 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.

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

COL-IV,1 originally isolated in an intact form from the Engelbreth-Holm-Swarm (EHS) tumor (1), is a large glycoprotein (Mr 500,000), which has the ability to polymerize into a network (2) on which other components such as laminin, entactin/nidogen, and heparan sulfate proteoglycan can bind and assemble (3). COL-IV also participates in the interaction of basement membranes with cells (4). This glycoprotein is composed of three chains ((alpha 1)2alpha 2), 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 alpha 1(IV) and 23 in the alpha 2(IV) chain. One interruption located at 100 nm from the amino end of the alpha 1(IV) has been made synthetically (peptide Hep-III) and was described by our group to mediate heparin and cell binding (5, 6). The sequence of this peptide (GEFYFDLRLKGDK) represents a pepsin-sensitive site (7, 8).

Collagens have been reported to interact with various cells at multiple interaction sites along the triple helix, in native or denatured form (9-11). The major collagen receptors have been demonstrated to belong to the beta 1 subgroup of the integrin family (12, 13), namely alpha 1beta 1, alpha 2beta 1, and alpha 3beta 1. In COL-IV, using isolated integrins and fragments of COL-IV, alpha 1beta 1 and alpha 2beta 1 integrin 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) (14). Further digestion of the CB3[IV] fragment by trypsin yields shorter triple helical fragments. Kern and co-workers (15), using solid-phase and inhibition assays demonstrated that alpha 1beta 1 and alpha 2beta 1 use two distinct binding sites located on two neighboring segments of COL-IV. The alpha 2beta 1 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 alpha 2 integrin binding site has been localized to the CB3 fragment of the alpha 1(I) collagen chain (16).

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 (17). 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 (17). In addition, various studies have localized the beta 1 family of integrins to the kidney (18) and to mesangial cells (19). The cell surface receptors alpha 1beta 1 and alpha 2beta 1 specifically mediate adhesion of cells to COL-IV (14).

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 beta 1 family of integrins was originally termed VLA or very late antigens and consists of at least six different members (18). The beta 1 integrins are important mediators of cell adhesion and migration; these receptors also influence cell proliferation, differentiation, and developmental processes (20-23). Additionally, integrins have a central role in extracellular matrix assembly (23, 24).

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 (25). Also, the same cell may simultaneously use different integrins to interact with the same matrix component (26). This redundancy is important for understanding the mechanism of ligand binding as well as the functional and signaling specificity of the various receptors (27).

We report here that a sequence contained in a synthetic peptide, HEP-III, positioned approx 100 nm from the amino-terminal end of the alpha 1(IV) contains a major alpha 2beta 1 integrin binding site of COL-IV. Monoclonal antibodies against the alpha 2 and beta 1 chains of the integrin family of receptors significantly inhibit mesangial cell attachment to this site in COL-IV, indicating that integrin alpha 2beta 1 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 alpha 1beta 1 and alpha 2beta 1 integrins.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cell Culture-- HMC were isolated from the kidneys of 19-22-week-old fetuses and characterized as described previously (28). The cells were cultured in Dulbecco's minimal essential medium (DMEM) supplemented with 20% fetal calf serum (Sigma), 15 mM HEPES, 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.

Isolation and Purification of Type IV Collagen and Type IV Collagen Domains-- COL-IV was isolated from (EHS) as described previously (29). 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 (30). The size of this fragment is approx 400 nm (30). This fragment also lacks some pepsin-sensitive interruptions in the triple helical domain, including the sequence contained in peptide Hep-III (31). 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 (29, 32). Peptide HEP-III was synthesized by the method of Barany and Merrifield (33) on a solid-phase support as described previously (5). Also, a solid-phase methodology was used for the synthesis of the triple helical polypeptide THP-III incorporating native sequences from the alpha 1(IV) chain (34).

Preparation of Anti-NC1 Antibodies and Their Fab Fragments-- Anti-NC1 antibodies were produced as described previously (29). 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.

Cell Adhesion to COL-IV and Fragments-- 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, approx 45-50% of each of these fragments adhere to plastic (5, 35). 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.

Inhibition of Cell Adhesion with Peptides-- 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.

mAbs to Integrin Receptors-- Mouse mAbs to the integrin receptors alpha 1 (SR84), alpha 2 (P1H5), alpha 3 (P3D11), alpha 4 (P4G9), alpha 5 (P3D10), and beta 1 (P5D2) have been previously described as adhesion-inhibiting antibodies in various systems (36, 37). 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.

Inhibition of Cell Adhesion with Monoclonal Antibodies-- Integrins alpha 1, alpha 2, alpha 3, alpha 5, and beta 1 have been previously reported to be present on mesangial cells (as determined by flow cytometry (38)). Subunits alpha 4 and alpha 6 were 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 alpha 1, alpha 2, and beta 1 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.

Determination of alpha 1beta 1 and alpha 2beta 1 Binding Sites on COL-IV-- 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 alpha 1, alpha 2, and beta 1 anti-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 (39). 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.

Affinity Chromatography-- HEP-III peptide was coupled to activated CH-Sepharose (Amersham Pharmacia Biotech) (40). 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 mM octyl-beta -D-glucopyranoside, 15 mM NaCl, 1 mM MgCl2, 1 mM MnCl2, 1 mM CaCl2, 1 mM N-ethylmaleimide, and 1 mM phenylmethylsulfonyl 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 (36). Elution fractions were electrophoresed by 7.5% SDS-polyacrylamide gel electrophoresis and analyzed by autoradiography.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Human Mesangial Cell Adhesion to COL-IV, Fragments, and Peptides-- 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. 1A). In similar experiments the pepsin-derived triple helical fragment bound a maximum of approx 65% cells, and NC1 bound approx 15% cells (Fig. 1A); HEP-III and THP-III peptides bound approx 15% cells (Fig. 1B). 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. 1C). 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 (approx 15%) as compared with the absence of specific antibodies to NC1 (Fig. 2).

                              
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Table I
Mesangial cell adhesion to COL-IV and fragments
This table summarizes the maximum adhesion seen of human mesangial cells to COL-IV, pepsin-derived fragment of COL-IV, which lacks the HEP-III domain, the major non-collagenous domain, and the HEP-III peptide, which is a sequence from one of the non-helical regions of the alpha 1 (IV) chain of COL-IV. Also, we have enumerated the maximum inhibition of binding of human mesangial cells to COL-IV and its fragments in the presence of monoclonal antibodies to the integrin subunits alpha 1, alpha 2, and beta 1.


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Fig. 1.   A, the binding of mesangial cells to COL-IV and its fragments was studied in a solid-phase assay. Concentrations of protein were converted from µg/ml to µM/ml so as to be able to compare percent binding between molecules. Radiolabeled mesangial cells bound to immobilized collagen IV (black-diamond ) to a maximum of approx 75%, the pepsin-derived triple helical fragment (bullet ) approx 65%, and the NC1 domain (open circle ) approx 15%. B, the binding of mesangial cells to peptide HEP-III and its triple helical counterpart THP-III. Radiolabeled mesangial cells were added to HEP-III (open circle ) and THP-III (bullet ) in solid phase and incubated for 30 min. Mesangial cells bound to both HEP-III and THP-III peptides in a concentration-dependent manner. C, inhibition of mesangial cell adhesion to solid-phase COL-IV by increasing concentrations of HEP-III and THP-III peptides. Radiolabeled mesangial cells were preincubated with the peptide inhibitors HEP-III (box-plus ) and THP-III (black-square) for 30 min and then added to COL-IV in 96-well plates for 30 min. Peptide concentration-dependent inhibition of binding was seen.


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Fig. 2.   Inhibition 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 alpha 1, alpha 2 and beta 1 subunits competed for the binding of mesangial cells to COL-IV. Antibodies to the integrin subunits alpha 3 and alpha 5, 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 approx 15% less than in the presence of control.

Inhibition of Binding in the Presence of Antibodies-- 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 (36, 37)) 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 alpha 1, alpha 2, alpha 3, alpha 4, alpha 5, and beta 1 anti-integrin antibodies, only antibodies against the alpha 1, alpha 2, and beta 1 competed for the binding of mesangial cells to COL-IV. The maximal observed inhibition in the presence of anti-beta 1 was approx 95%, in the presence of anti-alpha 1 it was approx 50%, and in the presence of anti-alpha 2 it was approx 70% (Fig. 2, Table I). Antibodies to alpha 1 and alpha 2 together caused inhibition to the same extent as beta 1. Antibodies to alpha 4 did not result in inhibition, as expected, since alpha 4 is not expressed by mesangial cells (38). Based on these assays, the alpha 1beta 1 and alpha 2beta 1 integrin receptors mediate the adhesion of human mesangial cells to COL-IV.

We further determined whether there was preferential use of alpha 1beta 1 or alpha 2beta 1 receptors 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-beta 1 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 beta 1 was almost complete on intact COL-IV. Only 30% inhibition was seen on peptide HEP-III (Fig. 3, A-C).


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Fig. 3.   Inhibition experiments in the presence of anti-integrin antibodies were performed on the pepsin-derived major triple helix (A), the major non-collagenous domain (B), and on the peptide HEP-III (C). Cells were incubated with solid-phase-immobilized COL-IV fragments and peptide HEP-III for a 30-min incubation. The following are the symbols representing the various antibodies in A, B, and C; square , HLA; bullet , alpha 1; black-square, alpha 2; open circle , alpha 1 and alpha 2; and black-diamond , beta 1.

Antibodies to the alpha 2 integrin subunit inhibited adhesion to triple helical fragments only minimally (approx 20%), even at the highest concentration, but effectively inhibited HMC adhesion to NC1 (approx 60% at the highest concentration) and HEP-III (approx 80% inhibition at the highest concentration) (Fig. 3, A-C).

Anti-alpha 1 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-alpha 1 and anti-alpha 2 were combined, inhibition of adhesion to all fragments was almost complete (Fig. 3, A-C).

Affinity Chromatography-- 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 alpha 1, alpha 2, alpha 3, beta 1 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-beta 1 integrin subunit mAb resulted in detection of 116- and 130-kDa proteins (Fig. 4). Similarly, immunoprecipitation of the same eluant with anti-alpha 2 and alpha 3 integrin subunit mAb resulted in detection of 116- and 130-kDa proteins (Fig. 4). The molecular masses correspond to the alpha 2, alpha 3 (130 kDa), and beta 1 (116 kDa) integrin subunits (36, 38). No proteins were seen where the alpha 1 subunit is expected (at approx 180 kDa) upon using the mAb against alpha 1 integrin subunits (Fig. 4).


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Fig. 4.   Immunoprecipitation analysis of human mesangial cell receptor proteins eluted from a HEP-III peptide affinity column. The proteins eluted by EDTA from the peptide column were immunoprecipitated with either anti-alpha 1, alpha 2, alpha 3, or beta 1 integrin subunit mAb. Proteins of 130, 130, and 116 kDa, corresponding to the alpha 2, alpha 3, and beta 1 integrin subunits, respectively, were immunoprecipitated. No proteins were immunoprecipitated where the alpha 1 subunit is expected (at approx 180 kDa).

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Type IV collagen has a role in ECM interaction with cells (4, 14, 18, 27, 41) in addition to providing mechanical stability by network formation (2, 30) and incorporation of other matrix components (3). The aim of our study was to identify the domains of COL-IV involved in HMC binding and also the cell surface receptor(s) involved in this interaction. COL-IV is found exclusively in basement membranes and in the intraglomerular basement membrane-like mesangial matrix. It differs from interstitial collagens structurally in possessing additional non-collagenous domains and interruptions of the Gly-X-Y repeat within its primary sequence (31). Assembly differs in the formation of a network (versus fibrils), which may be possible due to the flexibility of the molecules in the regions of non-helicity and interactions between the NC1 and collagenous domains (29).

We examined interactions between COL-IV and HMC, because the mesangial matrix surrounding HMC is enriched in COL-IV. Therefore, this glycoprotein should represent a major ligand for mesangial cells.

We had previously observed the following: (a) both alpha 2beta 1, a collagen receptor (12), and alpha 1beta 1, a receptor with affinity for COL-IV, COL-I, and laminin (42) were present on HMC as determined by flow cytometry and immunoprecipitation; and (b) alpha 1beta 1 and alpha 2beta 1 integrins were present in focal adhesions formed by HMC when these cells were seeded on COL-IV (38). These findings suggest that alpha 1beta 1 and alpha 2beta 1 contribute to mesangial cell adhesion to the mesangial matrix, which is composed mainly of COL-IV, and may modulate HMC-matrix interactions necessary for the functioning of this region.

In this report, we further examined HMC-COL-IV interactions to determine the domains of COL-IV involved in cell binding and to detect which integrin receptors participate in the interaction at each of these sites. We have presented evidence to indicate that intact COL-IV, pepsin-derived triple helical fragment (pepsin-IV), and NC1 domain each serve for HMC binding, albeit to a different extent. Under our experimental conditions, pepsin-IV was approximately four times more efficient than NC1 in promoting HMC adhesion. Nevertheless the NC1 domain sustained approx 15% HMC adhesion. We and others had previously reported that this main non-collagenous domain of COL-IV promotes the adhesion of human aortic endothelial and neuronal cells (35, 43). The contribution of the NC1 domain to HMC adhesion (to COL-IV) was additionally examined by the use of anti-NC1 antibodies in experiments which tested HMC binding to solid-phase-immobilized COL-IV. In these experiments, the presence of anti-NC1 antibodies resulted in approx 15% inhibition of HMC adhesion compared with the control, confirming the existence of cell binding activity of this domain, albeit to a lesser extent than the triple helical domain.

Additionally, peptide HEP-III, containing the sequence of an interruption of the Gly-X-Y motif in the alpha 1 chain of COL-IV molecule and its triple helical form were observed to sustain HMC adhesion. Inhibition of HMC binding to COL-IV in the presence of HEP-III and THP-III peptides confirms the function of this region in COL-IV adhesion to HMC. Even though the adhesion of HMC to solid-phase HEP-III and THP-III was significant but low compared with COL-IV, HEP-III and THP-III in solution almost completely inhibited adhesion to COL-IV. This may be attributed in part to the ability of HEP-III to initiate signal transduction pathways.2 The low percent adhesion of HMC may be explained by the low affinity of HEP-III as a cell-binding site, compared with COL-IV, or a lack of conformation of this peptide. This is supported by our finding that high peptide concentrations were required for both adhesion and inhibition of adhesion to COL-IV. It is also possible that large peptide concentrations were required for adhesion and inhibition of binding to COL-IV, because there are several other sites on COL-IV available for cell binding. Peptide Hep-III also promoted the adhesion of murine and human melanoma and aortic endothelial cells as well as heparin (5, 6, 44). A similar sharing of heparin and cell binding sites has been demonstrated in the long arm of laminin (45).

In summary, HMC adhesion was best sustained by intact and pepsin-treated COL-IV, which indicates one or more sites of high affinity binding to HMC; the main non-collagenous NC1 domain and a discontinuity of the triple helix represented in peptide Hep-III sustained HMC adhesion to a lesser extent, thus indicating sites of low affinity cell binding. However, low affinity interactions may be biologically relevant, since when combined these should increase the overall affinity of the binding.

We next examined which integrin subunits, from those present on HMC, mediate binding to COL-IV and its different domains. Competition experiments in which binding of HMC to COL-IV was studied in the presence of several anti-integrin antibodies demonstrated inhibition with antibodies to the alpha 2, alpha 1 and beta 1 integrin subunits indicating a role for both the alpha 1beta 1 and alpha 2beta 1 receptors in this interaction. Strong interactions in culture are mediated by focal adhesions (46). Focal adhesions are points of cell matrix contact responsible for attachment of cultured cells and the sites of initiation for actin containing stress fibers (47). Integrin receptors alpha 2beta 1 and alpha 1beta 1 have previously been demonstrated to bind collagen with the formation of focal adhesions (38, 47). It is therefore possible that in situ, HMC, which have several processes anchored in the mesangial matrix, use these two integrin receptors for attachment.

Our results indicate that both the NC1 and triple helical domains of COL-IV (pepsin-IV) bind to alpha 1beta 1 (Table I). Based on competition assays, the NC1 domain also interacts with alpha 2beta 1. The binding to pepsin-IV, which was previously reported by our group to lack the sequence contained in peptide HEP-III (5), was not inhibited significantly by anti-alpha 2 antibodies. The anti-alpha 2 subunit antibody inhibited adhesion of HMC to HEP-III to a large extent. This indicates that alpha 2beta 1 is the primary receptor involved in adhesion of this region of COL-IV to HMC. The anti-beta 1 antibody almost completely inhibited binding to COL-IV, pepsin-IV, and the NC1 domain but exhibited only partial inhibition on HEP-III. As described earlier HEP-III is a peptide derived from a region of interruption in the triple helical motif. Due to the high peptide concentration, it is very difficult to obtain complete inhibition using monoclonal antibodies. Also, the small peptide size makes it more accessible for integrin binding sites and thus more difficult to inhibit, especially compared with COL-IV. Affinity chromatography experiments using the HEP-III peptide as a matrix for binding labeled human mesangial cell lysate (Fig. 4) confirmed that beta 1 is the receptor used by human mesangial cells to bind HEP-III. These experiments demonstrated the presence of alpha 2beta 1 integrin and also alpha 3beta 1 integrin. This confirms the role of alpha 2beta 1 in binding to HEP-III as demonstrated by inhibition of adhesion.

The binding of alpha 3beta 1 integrin to Hep-III by affinity chromatography deserves noting. Anti-alpha 3 antibodies did not inhibit HMC adhesion to COL-IV even though the alpha 3 was present on HMC as we reported previously (38) (Fig. 2). The presence of alpha 3beta 1 on the Hep-III affinity column can be explained by a low affinity interaction between alpha 3beta 1 and HEP-III, probably as a postadhesion process. This possibility is strengthened by the data of DiPersio and co-workers (48), who have observed a postadhesion recruiting of integrin alpha 3beta 1 into focal contacts.

It has been reported that alpha 1beta 1 integrin binds preferentially to COL-IV and has lower affinity for type I collagen (42) as well as for laminin (49, 50). The alpha 2beta 1 integrin also binds collagens and laminin (12, 51). Both alpha 1beta 1 and alpha 2beta 1 integrins are present on human and rat mesangial cells in culture (52, 53) and in situ (38, 41, 54).

Kern and co-workers (15) and Eble et al. (55) have studied the adhesion of isolated integrins alpha 1beta 1 and alpha 2beta 1 to a cyanogen bromide-derived fragment CB3(IV) of COL-IV, which is located 100 nm from the amino terminus of the molecule. This fragment was demonstrated to contain sites for both alpha 1beta 1 and alpha 2beta 1 integrins. Upon trypsin digestion of CB3(IV) fragments F1-F4 were obtained. Fragment F1 (alpha 1(IV) positions 376-558 and alpha 2(IV) positions 406-554) was seen also to contain sites of binding of alpha 1beta 1 and alpha 2beta 1. However, fragment F4 (alpha 1(IV) positions 430-520 and alpha 2(IV) positions 433-516) bound only to alpha 1beta 1 and not at all to alpha 2beta 1. Since fragment F4 is missing the amino acids 531-543, which make up the peptide HEP-III, these data corroborate our results that adhesion to HEP-III is mediated only by integrin alpha 2beta 1 and not by alpha 1beta 1.

In summary, human mesangial cells bind COL-IV avidly using cell surface receptors of the integrin family, namely alpha 1beta 1 and alpha 2beta 1. Apparently, COL-IV has several sites for cellular interaction along its triple helix, at least one interruption, and the main non-collagenous NC1 domain. In addition to peptide HEP-III, with adhesion mediated by alpha 2beta 1 of HMC, at least two other binding sites for mesangial cells were found: one or more in the NC1 domain primarily mediated by alpha 1beta 1 along with alpha 2beta 1 and on the triple helical fragment lacking the HEP-III sequence, mediated mainly by the alpha 1beta 1 integrin receptor. Our data indicate that there is more than one integrin receptor for various domains of type IV collagen on human mesangial cells and more than one site of interaction with this complex basement membrane glycoprotein. Binding at different sites suggests involvement in different cellular functions and could be associated with the activation of different cell signaling mechanisms.

    ACKNOWLEDGEMENT

We thank Kimberley Pinkham for expert technical help in the isolation of fetal mesangial cells.

    FOOTNOTES

* This work was supported by National Institutes of Health NIDDK Grants 39216 and 43574 (to E. C. T.) and 44494 (to G. B. F.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom all correspondence should be addressed: Box 609 UMHC, Dept. of Laboratory Medicine and Pathology, University of Minnesota Medical School, 420 Delaware St. S.E., Minneapolis, MN 55455. Tel.: 612-625-9171; Fax: 612-625-0617.

1 The 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.

2 G. B. Fields, unpublished observations.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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