From the Departments of Laboratory Medicine and
Pathology and ¶ Pediatrics, School of Medicine, University of
Minnesota, Minneapolis, Minnesota 55455,
Institute of Biology, NRCPS Demokritos,
Athens, Greece, ** Fred Hutchinson Cancer Center, Seattle,
Washington, and
Department of Biological Sciences, University of
California, Santa Barbara, California
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ABSTRACT |
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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
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-
1 antibodies inhibited the binding
of HMC to COL-IV, the NC1, and triple helical domains, but not to peptide HEP-III. Anti-
1 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
2
1
along with
3
1. We conclude that
2
1 and
1
1
integrins mediate HMC adhesion to COL-IV. Peptide HEP-III is a major,
specific site for
2 integrin-mediated binding of
mesangial cells to COL-IV. Both the
1
1
and
2
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.
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INTRODUCTION |
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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 ((1)2
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
1(IV) and 23 in
the
2(IV) chain. One interruption located at 100 nm from
the amino end of the
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 1 subgroup of the integrin family (12, 13),
namely
1
1,
2
1, and
3
1.
In COL-IV, using isolated integrins and fragments of COL-IV,
1
1 and
2
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
1
1 and
2
1
use two distinct binding sites located on two neighboring segments of
COL-IV. The
2
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
2 integrin binding site has been
localized to the CB3 fragment of the
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 1 family of integrins to the kidney
(18) and to mesangial cells (19). The cell surface receptors
1
1 and
2
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 1 family of integrins was
originally termed VLA or very late antigens and consists of at least
six different members (18). The
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 100 nm from the amino-terminal end of the
1(IV) contains a major
2
1
integrin binding site of COL-IV. Monoclonal antibodies against the
2 and
1 chains of the integrin family of
receptors significantly inhibit mesangial cell attachment to this site
in COL-IV, indicating that integrin
2
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
1
1 and
2
1
integrins.
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MATERIALS AND METHODS |
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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 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
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, 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 1 (SR84),
2 (P1H5),
3 (P3D11),
4 (P4G9),
5
(P3D10), and
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 1,
2,
3,
5, and
1 have been
previously reported to be present on mesangial cells (as determined by
flow cytometry (38)). Subunits
4 and
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
1,
2, and
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%.
Determination of 1
1 and
2
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
1,
2, and
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--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.
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RESULTS |
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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 65%
cells, and NC1 bound
15% cells (Fig. 1A); HEP-III and
THP-III peptides bound
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 (
15%) as compared with the absence of specific antibodies to NC1 (Fig. 2).
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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 1,
2,
3,
4,
5,
and
1 anti-integrin antibodies, only antibodies against
the
1,
2, and
1 competed
for the binding of mesangial cells to COL-IV. The maximal observed
inhibition in the presence of anti-
1 was
95%, in the
presence of anti-
1 it was
50%, and in the presence
of anti-
2 it was
70% (Fig. 2, Table I). Antibodies
to
1 and
2 together caused inhibition to
the same extent as
1. Antibodies to
4 did
not result in inhibition, as expected, since
4 is not
expressed by mesangial cells (38). Based on these assays, the
1
1 and
2
1
integrin receptors mediate the adhesion of human mesangial cells to
COL-IV.
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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 1,
2,
3,
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-
1
integrin subunit mAb resulted in detection of 116- and 130-kDa proteins
(Fig. 4). Similarly, immunoprecipitation of the same eluant with anti-
2 and
3
integrin subunit mAb resulted in detection of 116- and 130-kDa proteins
(Fig. 4). The molecular masses correspond to the
2,
3 (130 kDa), and
1 (116 kDa) integrin subunits (36, 38). No proteins were seen where the
1
subunit is expected (at
180 kDa) upon using the mAb against
1 integrin subunits (Fig. 4).
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DISCUSSION |
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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
2
1, a collagen receptor (12), and
1
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)
1
1 and
2
1 integrins were present in focal adhesions formed by HMC when these cells were seeded on COL-IV (38). These findings suggest that
1
1 and
2
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 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
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
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 2,
1 and
1 integrin subunits indicating a role for both the
1
1 and
2
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
2
1 and
1
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 1
1 (Table I).
Based on competition assays, the NC1 domain also interacts with
2
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-
2 antibodies. The anti-
2 subunit
antibody inhibited adhesion of HMC to HEP-III to a large extent. This
indicates that
2
1 is the primary receptor
involved in adhesion of this region of COL-IV to HMC. The
anti-
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
1 is the receptor used by human mesangial
cells to bind HEP-III. These experiments demonstrated the presence of
2
1 integrin and also
3
1 integrin. This confirms the role of
2
1 in binding to HEP-III as demonstrated
by inhibition of adhesion.
The binding of 3
1 integrin to Hep-III by
affinity chromatography deserves noting. Anti-
3
antibodies did not inhibit HMC adhesion to COL-IV even though the
3 was present on HMC as we reported previously (38)
(Fig. 2). The presence of
3
1 on the Hep-III affinity column can be explained by a low affinity interaction between
3
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
3
1 into focal
contacts.
It has been reported that 1
1 integrin
binds preferentially to COL-IV and has lower affinity for type I
collagen (42) as well as for laminin (49, 50). The
2
1 integrin also binds collagens and
laminin (12, 51). Both
1
1 and
2
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 1
1 and
2
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
1
1 and
2
1
integrins. Upon trypsin digestion of CB3(IV) fragments F1-F4 were
obtained. Fragment F1 (
1(IV) positions 376-558 and
2(IV) positions 406-554) was seen also to contain sites
of binding of
1
1 and
2
1. However, fragment F4
(
1(IV) positions 430-520 and
2(IV)
positions 433-516) bound only to
1
1 and
not at all to
2
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
2
1 and not by
1
1.
In summary, human mesangial cells bind COL-IV avidly using cell surface
receptors of the integrin family, namely
1
1 and
2
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
2
1 of HMC, at least two other binding
sites for mesangial cells were found: one or more in the NC1 domain primarily mediated by
1
1 along with
2
1 and on the triple helical fragment
lacking the HEP-III sequence, mediated mainly by the
1
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.
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ACKNOWLEDGEMENT |
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We thank Kimberley Pinkham for expert technical help in the isolation of fetal mesangial cells.
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FOOTNOTES |
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* 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.
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REFERENCES |
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