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INTRODUCTION |
EMILIN1-1 (1)
is the prototype of a new family of glycoproteins, EMILINs (2-5),
which are expressed in a tissue-specific and developmentally regulated
manner and whose biological activities are still to be defined
properly. EMILIN-1 is composed of a cysteine-rich domain (EMI domain)
at the N terminus (6), a long segment of about 650 residues with a high
potential for forming coiled-coil helices, a short uninterrupted
collagenous stalk, and a C1q-like globular domain at the C terminus
(gC1q-l), representing a structurally unique component. The gC1q-1
domain is necessary for the noncovalent formation of homotrimers that
are then linked by disulfide bonds giving rise to very large
extracellular aggregates (7). EMILINs are members of the large
C1q/tumor necrosis factor superfamily of proteins that are
characterized by the presence of a gC1q domain; the superfamily (1)
includes several collagens among which types VIII and X, the
recognition component of the classical complement pathway C1q-C', and AdipoQ.
Elastic fibers and associated fibrillin-containing microfibrils (8) are
important structural components of the extracellular matrix (ECM) of
most connective tissues. EMILIN-1 forms a fibrillar network in
vitro and in the ECM of several tissues including blood vessels,
skin, heart, lung, kidney, and cornea (9-13). This glycoprotein codistributes with elastin in most sites and likely constitutes an
associated component of elastic fibers. EMILIN-1 is localized mainly at
the interface between amorphous elastin and the surrounding microfibrils, and it has been implicated in the correct deposition of
elastin in vitro (14). In addition, EMILIN-1-reacting
structures were often observed in vivo closely adjacent to
the surface of cells (15, 16), and thus it seemed likely that EMILIN-1
could also interact directly with cell membrane receptors. The
constituents of elastic fibers and microfibrils not only display a
repertoire of multiple interactions but are recognized by integrin cell
surface receptors. Integrins are widely distributed heterodimeric
glycoproteins that play a fundamental role in cell adhesion and
migration, in cell differentiation, and in many different biological
processes. In particular the integrin
v
3,
a promiscuous RGD-dependent integrin (17), was shown to
mediate adhesion and spreading of many cell types on elastic
fiber-associated constituents such as fibrillin-1 and -2, MAGP-2, and
fibulin-5 (18-22). EMILIN-1 does not contain RGD motifs (1), but in
preliminary studies it was shown to behave as a ligand for some tumor
cells (1, 2), although the receptors involved had not been investigated.
In this study with the use of purified EMILIN-1 and its fragments we
addressed the adhesive function of EMILIN-1 and pinpointed the gC1q-1
domain as the major and sufficient domain responsible for cell
adhesion. Our results suggest that the gC1q-1 domain of EMILIN-1, in
addition to its proven function in the initial steps of the assembly of
EMILIN-1 homotrimers and multimers (7/12), has a novel cell
adhesive role and highlight that gC1q-1 is the first globular C1q
domain that is a ligand for a
1 integrin, namely
4
1.
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EXPERIMENTAL PROCEDURES |
Materials--
Function-blocking anti-integrin monoclonal
antibodies (mAbs) were obtained as follows. Anti-
2
(clone 12171) was from Virgil Woods (Lombardi Cancer Research Center,
Georgetown University, Washington, D. C.). Anti-
3 clone
F4 was from Luciano Zardi (Istituto Tumori Genova, Italy).
Anti-
l (clone FB12), anti-
3 (clone P1B5), anti-
4 (clones P1H4 and P4G9),
anti-
5
1 (clone JBS5),
anti-
9 (clone Y9A2), and
anti-
v
3 (clone LM 609) were from Chemicon (Chemicon International Inc., Temecula, CA). Anti-
v was
from Robert Pytela (Section for Cardiovascular Research, University of
California, San Francisco). Anti-
6 (clone GoH3) was from
Arnoud Sonnenberg (Division of Cell Biology, The Netherlands Cancer
Institute, Amsterdam). Anti-
1 (clone 4B4) was from
Coulter/Immunotech S.A., Marseille, France. The mAb PS2 against the
murine
4 chain was from ATCC (Rockville, MD).
Anti-paxillin mAb was purchased from Chemicon.
The human SW982 (synovial sarcoma), Hs913T, and HT-1080
(fibrosarcoma), SK-UT-1 (leiomyosarcoma), Jurkat and RAMOS
(lymphoblastoid T cell), K562 (myeloid leukemia) cell lines, and the
murine NIH3T3 (fibroblast) cells were purchased from ATCC. FLG 29.1 cells have been described previously (23) and were obtained from Dr. V. Gattei (CRO-IRCCS, Aviano, Italy). The murine NQ22 and NQ29
(lymphoblastoid T cells) cell lines have been described (24). Uterine
smooth muscle cells were purchased from Biowittaker (Walkersville, MD). The cell lines were maintained in Dulbecco's medium containing 10%
fetal calf serum. K562 cells stably transfected with the
4 integrin chain (
4/K562) were obtained
from Dr. Joaquin Teixido (Centro de Investigaciones Biologicas,
Department of Immunology, Madrid, Spain) and maintained in RPMI medium
containing 10% fetal calf serum and 1 mg/ml G418. Before use in
functional assays expression of
4 was evaluated by flow
cytometry with specific anti-
4 mAbs, and the cells were
found to be more than 80% positive. The C1q component of human
complement was purchased from Sigma, and plasma fibronectin (FN) was
from Calbiochem-Novabiochem. Vitronectin (VN) was purified from human
plasma as described by Yatohgo et al. (25). Integrins
purified from human placenta were purchased from Chemicon
(
1
1) or obtained from Dr. Martin J. Humphries (Wellcome Trust Centre for Cell-Matrix Research, University
of Manchester, U. K.) (
4
1).
Flow Cytometry--
Expression of various cell surface receptors
was analyzed by single-color direct or indirect immunofluorescence by
utilizing mAbs recognizing the following integrin chains:
1, CD29;
2, CD49b;
3,
CD49c;
4, CD49d;
5, CD49e;
6, CD49f;
3, CD61 (Coulter/Immunotech S.A). As second step reagents, isotype-matched control mAbs and phycoerythrin-conjugated F(ab')2 fragments of goat
anti-mouse Igs (Jackson Immunoresearch Laboratories, West Grove, PA)
were used. Viable, antibody-labeled cells were identified according to
their forward and side scatter, electronically gated, and assayed for
surface fluorescence on a FACScan flow cytometer (Becton-Dickinson Immunocytometry System, San Jose, CA).
Production of Recombinant EMILIN-1 Polypeptides and Protein
Purification--
293 cells, constitutively expressing the EBNA-1
protein (293-EBNA), were transfected with the constructs for EMILIN-1
or the LEU-COL-gC1q-1 as described previously (12). The cells expanded to mass culture and were then maintained for 2 days in serum-free medium to allow accumulation of the polypeptides in the cell
supernatant. Partial purification was achieved by dialysis of the
conditioned medium at 4 °C against 0. 1 M NaCl, 20 mM Tris-HCI, pH 6.8. A further purification step was
achieved by chromatography on a DEAE-cellulose column (Amersham
Biosciences) equilibrated in the same buffer. The bound material was
eluted with a NaCl gradient in 20 mM Tris-HCl, pH 6.8. Then
the peak fractions containing the LEU-COL-gC1q-1 polypeptide were
pooled, dialyzed against 50 mM Tris-HCl, 1.2 M
ammonium sulfate, pH 8.8, and loaded onto a column of phenyl-Sepharose
CL-4B (Amersham Biosciences); bound material was eluted with a linear
gradient of ammonium sulfate in 50 mM Tris-HCl, pH 8.8. The
pooled fractions containing EMILIN-1 and obtained from the elution of
the DEAE-cellulose column were further purified by size exclusion
chromatography using Sepharose CL-4B (1.0 × 90.0-cm column;
Amersham Biosciences).
The sequence corresponding to the C-terminal domain of EMILIN-1
(gC1q-1) was amplified by reverse transcription-PCR from human aorta
mRNA and was then ligated in-frame in the His6-tagged
pQE30 expression vector (Qiagen GmbH, Germany). 500 ml of liquid
culture grown at 0.6 A600 nm was induced with 2 mM isopropyl-1-thio-
-D-galactopyranoside for
3 h at 37 °C. The culture was then centrifuged at 4,000 × g for 20 min, and the cell pellet was resuspended in
sonication buffer (50 mM sodium phosphate, 0.3 M NaCl, pH 8.0) at 5 volumes/g, wet weight. The sample was
frozen in a dry ice/ethanol bath, thawed in cold water, and sonicated
on ice (1-min bursts/1-min cooling, 2-300 watts), and cell breakage
was monitored by measuring the release of nucleic acids at
A600 nm. The cell lysate was centrifuged at
10,000 × g for 20 min, the supernatant was collected, and purification of the His6-tagged recombinant
fragment was performed by affinity chromatography on
nickel-nitrilotriacetic acid resin (Qiagen GmbH) under native
conditions. The recombinant protein was eluted from the affinity column
in sonication buffer, pH 6.0, containing 10% glycerol and 0.2 M imidazole, and the native status of gC1q-1 was checked by
circular dichroism. The EMILIN-1 proteins that were used in the present
study, including the recombinant human full-length EMILIN-1 and the
LEU-COL-gC1q-1 polypeptide produced in 293-EBNA cells as well as the
gC1q-1 polypeptide produced in Escherichia coli cells (Fig.
1A), were analyzed by SDS-PAGE under reducing conditions using 8% gels and Coomassie Brilliant Blue
staining and are shown in Fig. 1B.

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Fig. 1.
Schematic diagram of the different constructs
of human EMILIN-1. A, the various polypeptides were
produced in 293-EBNA cells using the pCEP-Pu/AC7 vector (EMILIN-1,
LEU-COL-gC1q-1) or in E. coli using the pQE30 vector
(gC1q-1). B, relative migration on SDS-PAGE of recombinant
EMILIN-1 (lane 1), LEU-COL-gC1q-1 (lane 2), and
gC1q-1 (lane 3). Polypeptides collected from serum-free
culture medium of 293-EBNA cells or bacteria were separated by 10%
SDS-PAGE under reducing conditions and immunoblotted with a polyclonal
antibody against recombinant EMILIN-1.
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Production of mAbs--
BALB/c mice were immunized with EMILIN-1
or with the recombinant gC1q-1 polypeptide. 0.1 mg of recombinant
protein for each mouse was emulsified with complete Freund's adjuvant
and injected intraperitoneally. Four repeated injections every 10-14
days with the same amount of protein emulsified with incomplete
Freund's adjuvant were administered. Three days after the last booster injection, the spleens were removed and the splenocytes fused with the
cell line P3X63Ag8/NS-1 (26). Culture fluids of the resulting
hybridomas were screened for anti-EMILIN-1 activity in ELISA and
Western blotting. Several hybridomas that recognized EMILIN-1 and
reacted with the antigens in ELISAs were selected and subcloned twice
before using. Rabbit antiserum against human EMILIN-1 obtained by
immunization with purified recombinant protein was absorbed onto a
CNBr-activated Sepharose resin saturated with mock-transfected cell
proteins as described before (1).
Competitive ELISA--
Polystyrene wells coated with gC1q-1 and
saturated with 2% (w/v) BSA were first incubated for 1 h with
3-fold dilutions of unlabeled mAbs against EMILIN-1 purified by protein
A-Sepharose (Amersham Biosciences) followed by a 1-h incubation with
15-20 ng of different biotin-labeled mAbs. Purified mAbs were labeled by the biotin procedure as recommended by the manufacturer (Pierce). 100% binding was calculated as the binding obtained in the presence of
an unrelated mAb as inhibitor.
Cell Adhesion Assay--
The quantitative cell adhesion assay
used in this study is based on centrifugation and has been described
previously (27, 28). Briefly, six-well strips of flexible polyvinyl
chloride denoted CAFCA (centrifugal assay for fluorescence-based cell
adhesion) miniplates, covered with double-sided tape (bottom units),
were coated with the different substrates. Cells were labeled with the
vital fluorochrome calcein AM (Molecular Probes) for 15 min at
37 °C, rinsed extensively in Ca2+/Mg2+-free
PBS, followed by one rinse in PBS containing 1 mM EDTA, and
then aliquoted into the bottom CAFCA miniplates at a density of 20 to
50 × 104 cells/ml. Cell adhesion to substrates was
assayed in PBS containing 0.5% polyvinylpyrrolidone (PVP 360, Sigma),
1 mM MgC12, and 1 mM/liter
CaC12, and 2% India ink as a fluorescence quencher. CAFCA miniplates were centrifuged at 142 × g for 5 min at
37 °C to synchronize the contact of the cells with the substrate.
The miniplates were then incubated for 20 min at 37 °C and
subsequently mounted together with a similar CAFCA miniplate to create
communicating chambers for subsequent reverse centrifugation. In some
instances, cells were preincubated with blocking antibodies or
nonblocking control antibodies for 30 min and then added to the
miniplates coated with the various substrates. Another series of
experiments, in which the CAFCA miniplate assemblies were centrifuged
at 43, 170, and 380 × g, were also performed with the
aim of determining the relative strength of cell adhesion to the
various substrates. The relative number of cells bound to the substrate
(i.e. remaining in the wells of the bottom miniplates) and
cells that fall to bind to the substrate (i.e. remaining in
the wells of the top miniplates) was estimated by top/bottom
fluorescence detection in a computer-interfaced SPECTRAFluor
Plus microplate fluorometer (TECAN). Fluorescence values were
elaborated by the CAFCA software (TECAN) to determine the percentage
adherent cells, of the total cell population analyzed, according to a
previously published formula (28, 29). Statistical significance
determined by Student's t test was set at p < 0.001. In experiments aimed at examining the effects of blocking
antibodies, the various antibodies were added directly to the wells,
just before plating the cells. In other experiments aimed at examining
the effect of inhibition of cell adhesion by purified integrins,
soluble
1
1 or
4
1 was added (5 µg/ml directly to the
wells, just before plating the cells).
Immunofluorescence--
Multiwell plates or acid-washed
coverslips were coated with 10 µg/ml FN, 10 µg/ml EMILIN-1, 20 µg/m1 gC1q-1, 10 µg/ml VN, 20 µg/ml C1q-C', and the 20 µg/ml
synthetic peptide CS-1 for 16 h at 4 °C, and nonspecific
binding was blocked with 1.0% radioimmunoassay grade BSA (Sigma).
Cells were then plated onto the various substrates for 30-40 min as
indicated, then they were fixed with 4% (w/v) formaldehyde for 10 min
and permeabilized in PBS containing 0.1% Triton X-100 for 2 min. The
cells were then incubated for 1 h with primary antibodies in PBS,
washed, and incubated further with the appropriate secondary antibodies
and with 2 µg/ml Texas Red-conjugated phalloidin (Molecular Probes)
for 1 h. After extensive washes, coverslips were mounted in Mowiol
4-88 (Calbiochem-Novabiochem) containing 2.5% (w/v) DABCO (Sigma).
Images were acquired with a Bio-Rad MRC-1024 confocal system using
Bio-Rad Lasersharp software and a 60× phase/fluorescence objective on
a Diaphot 200 Nikon.
Motility Assay--
Migration experiments involving haptotactic
movement of the cells through a porous membrane were performed using
Transwells (Corning Costar Corporation, Cambridge, MA). The underside
of the insert membrane was coated with the various molecules in
bicarbonate buffer at 4 °C overnight and blocked with 1% BSA for
1 h at room temperature. Cells were aliquoted into the upperside
of each insert unit (1 × 105 cells/insert), and after
4 h the number of migrated cells/field was evaluated under the microscope.
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RESULTS |
Cell Adhesion--
The potential cell adhesive capacity of
recombinant EMILIN-1 had been already assessed using several cell lines
and found to be of comparable potency but of distinct pattern with that of FN, a prototype adhesive molecule (2). To investigate further the
site(s) of cell attachment, mAbs against the full sized recombinant EMILIN-1 or the gC1q-1 domain were generated and used in conjunction with recombinant proteins to localize the reactive epitopes by solid
phase ELISA. The localization of the antibody epitopes is shown
schematically in Fig. 2A. MAb
837C was the only one that bound to the full sized EMILIN-1 and
not to the other polypeptides, indicating that its epitope maps to the
coiled-coil or the EMI domain region. Instead, several mAbs bound to
both EMILIN-1 and the LEU-COL-gC1q-1 polypeptide, indicating that they
map in the COL domain or in the short upstream region. Two mAbs (2F1
and 2C8) bound only to the LEU-COL-gC1q-1 polypeptide, suggesting that
their epitopes are masked in full sized EMILIN-1 during its assembly
process. Finally, seven mAbs recognized all three polypeptides and thus
map to the gC1q-1 domain.

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Fig. 2.
Perturbation of cell attachment by
anti-EMILIN-1 monoclonal antibodies. A, schematic
diagram depicting the epitope localization of anti EMILIN mAbs. The
presumed localization is based on solid phase ELISAs with recombinant
proteins. B, perturbation of cell attachment to EMILIN-1.
Wells coated with 10 µg/ml EMILIN-1 were preincubated with the
indicated antibodies for 1 h at 37 °C, and the cells were
allowed to adhere at 37 °C in the presence of 1 mM
Ca2+ or 1-3 mM Mg2+. C,
the epitope recognized by the function-blocking mAb 1H2 is unique. The
1H2 mAb was biotin labeled and added to wells coated with EMILIN-1 in
the presence or absence of various anti EMILIN-1 mAbs.
Unrelated represents the number of cells remaining attached
in the presence of an unrelated mAb.
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Next, to determine which domain(s) would be necessary and sufficient to
promote cell adhesion, cells were allowed to adhere to EMILIN-1 in the
presence of mAbs directed against different epitopes of the EMILIN-1
molecule. Although the presence of antibody 1H2 (mapping to the gC1q-1
domain) caused complete inhibition of SW982 cell attachment (Fig.
2B), mAbs mapping to other domains were totally ineffective.
Also, several mAbs mapping to the gC1q-1 domain were unable
to block cell adhesion. Because by increasing the amounts of
nonblocking antibodies added we could not obtain lower attachment
levels (data not shown), it seems reasonable to conclude that blocking
by 1H2 was highly specific. To investigate further the binding site
specificity of mAb 1H2 on the gC1q-1 domain a competitive inhibition
solid phase ELISA binding assay was performed using EMILIN-1 as a
substrate and a number of mAbs as competitors. Binding of
biotin-labeled 1H2 to EMILIN-1 was unaffected by several mAbs mapping
on the gC1q-1 domain except 1H2 itself (Fig. 2C), suggesting
that this mAb detects a unique epitope involved in cell recognition and adhesion.
Characterization of Cell Adhesion to EMILIN-1 and
gC1q-1--
Although EMILIN-1 promoted an effective cell
adhesion, the very large disulfide-bonded aggregates of this protein
produced by transfected 293-EBNA cells make it less suitable than
gC1q-1 to investigate its role in cell adhesion phenomena further. As a
preliminary analysis we sought then to compare the adhesive function of
full sized EMILIN-1 with that of the gC1q-1 polypeptide on a larger
spectrum of cells (Table I). The two
ligands displayed a comparable ability to promote cell adhesion, with
EMILIN-1 displaying a somewhat higher percentage of cell attachment.
Only HT-1080 cells were found not to adhere to both ligands to the same
extent, but clearly preferred EMILIN-1 (97% adhesion on EMILIN-1
versus 45% on gC1q-1).
Next, the kinetics of cell adhesion was investigated. Cell attachment
on both EMILIN-1 and gC1q-1 showed a kinetics similar to that on FN, as
50% cell binding was observed within 10 min of plating, and the
maximum level of attachment was reached with all ligands at 20 min
(Fig. 3A). Furthermore, SW982
cells attached to both EMILIN-1 and gC1q-1 in a
dose-dependent manner (Fig. 3B). Under the
conditions used, adhesion reached plateau levels at a coating
concentration of about 5 µg/ml for both substrates, although on a
molar base full sized EMILIN-1 resulted at least 10 times more
effective than the isolated gC1q-1 domain. However, the coating
efficiency of the two ligands was verified to be similar on the type of
plastic used for the CAFCA (data not shown). Divalent cations such as
Ca2+ and Mg2+ are generally necessary for
integrin-ligand recognition (29). To evaluate the integrin dependence
of the EMILIN-1 and gC1q-1 adhesive activities, cell adhesion was
determined in the absence of cations and found to be negligible (data
not shown). Furthermore, cations regulated cell adhesion in a
concentration-dependent manner, with Mg2+ showing a
higher effect (data not shown).

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Fig. 3.
Cell attachment to EMILIN-1 and gC1q-1.
Kinetics of cell attachment (A) and dose-response
relationship (B) are shown. Recombinant gC1q (open
circles) was purified under conditions favoring refolding and
plated at the indicated concentrations. Recombinant EMILIN-1
(closed circles) was purified from the cell culture medium
of 293-EBNA cells. BSA (open squares) and human FN
(closed squares) were used as negative and positive control
ligands. Bars represent the S.D. of triplicate assays.
C, assessment of the relative strength of cell attachment to
EMILIN-1, gC1q-1, and FN by varying the centrifugal force applied to
dislodge the cells. SW982 cells were allowed to attach to the
substrates coated with the various ligands at 3 µg/ml, in the
presence of 1 mM Ca2+ or 1-3 mM
Mg2+.
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The cell adhesion assay used in this study permits the estimation of
the relative adhesion strength displayed to a given substrate by
varying the cell detachment force applied. Thus, we next examined the
profile of cell attachment to EMILIN-1 and gC1q-1 compared with the
adhesion strength obtained on FN (Fig. 3C). The force necessary to detach 50% SW982 cells was
500 × g,
250 × g, and 270 × g for FN,
EMILIN-1, and gC1q-1, respectively. Taken together these results
indicate that the constitutively active receptors involved confer to
these cells the capability of recognizing EMILIN-1 and gC1q-1 with a
similar but lower relative avidity compared with the levels attained
with FN.
Cell Adhesion to gC1q-1 Is
1
Integrin-dependent--
The above results suggested the
potential for integrin-gC1q-1 interactions. In fact, consistent with
the inhibition of cell attachment by EDTA and with the cation
dependence of cell attachment (data not shown), the constitutive
attachment of SW982 cells to gC1q-1 but also to EMILIN-1 was entirely
mediated by a
l subunit-containing integrin (Fig.
4). In fact, cell adhesion was completely
blocked by 4B4, a neutralizing
1 integrin subunit mAb,
whereas the
v
3-blocking mAb failed to
inhibit adhesion.

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Fig. 4.
The function-blocking
1 mAb perturbs cell attachment to
EMILIN-1 and gC1q-1 but not to C1q-C'. The miniplate wells were
coated with 10 µg/ml EMILIN-1, gC1q-1, C1q-C', or VN. The antibodies
were added at 5 µg/ml just before cell plating. Results shown are the
average ± S.D. of triplicate experiments.
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C1q-C' has been shown to support adhesion and spreading of endothelial
cells (30, 31), to induce chemotaxis and chemokinesis of mast cells
(32), and to promote eosinophil migration (33). Cell adhesion to C1q-C'
could be blocked by RGD-containing peptides (34), supporting the notion
that C1q-C'-mediated adhesion and spreading may involve, in addition to
the various nonintegrin C1q receptor(s) (35), the participation of
integrin(s). Whether C1q-C' promoted cell adhesion in our assay system
and whether this activity could be abolished by the 4B4
1-blocking mAb were then investigated in analogy with
the present findings with both EMILIN-1 and gC1q-1. C1q-C' strongly
promoted cell adhesion of SW982 cells; however, treatment with 4B4 mAb
was totally ineffective (Fig. 4). As expected, this mAb displayed no
inhibition of the
v
3-dependent VN-mediated cell adhesion.
Cell Adhesion to EMILIN-1/gC1q-1 Is
4
1-dependent--
Because the
results described above indicate that SW982 cells bound to EMILIN-1 or
gC1q-1 domain via a
1 integrin, we next examined the
effects of
subunit function-blocking mAbs in inhibiting the gC1q-1
or the EMILIN-1 binding activity. Treatment of cells with inhibitory
antibodies indicated that the
subunit was neither
1,
2,
3,
5,
6,
9, nor
v because blocking mAbs to these subunits had no effect on cell attachment. In separate experiments their function blocking activity for their respective ligands, i.e. FN, VN, and LNs, was confirmed with various cell
lines (data not shown). In contrast, only the anti-
4 mAb
P1H4 fully abrogated cell adhesion to gC1q-1 and up to 70% to
EMILIN-1. SW982 cells express consistent levels of
4
integrin chains as well as
1,
3,
5,
v, and
1 as evaluated
by fluorescence-activated cell sorter analysis (data not shown). That
4
1 really participates in cell adhesion
to gC1q-1 and the inhibition detected is not the mere consequence of
the expression levels of this integrin were demonstrated also by the
dose dependence of inhibition of adhesion (Fig.
5) and by the finding that a control
anti-
4 mAb, P4G9, directed against epitope A (not
related to cell attachment to ECM) on the
chain (36), was
ineffective (Fig. 5A) as was the function-blocking mAb PS2
recognizing the murine
4 (data not shown). To
investigate further the specificity of the recognition by
4
1 integrin, additional experiments were
performed. Binding between EMILIN-1 and
4
1 integrin was evaluated in a solid
phase assay (Fig. 5C, inset). Binding to
4
1 was twice as high as that to
1
1, and it was specifically inhibited by
anti-
4 function-blocking mAb. Second, EMILIN-1 and FN
were adsorbed on the plastic substrate, and cell adhesion was evaluated
in the presence or in the absence of purified
1
1 or
4
1
integrins. Although
1
1 had no effect, a
40% reduction in cell adhesion was detected only when soluble
4
1 was added (Fig. 5C). As a
third approach, adhesion of
4/K562 cells to EMILIN-1 was
analyzed. Adhesion to EMILIN-1 reached 45% (Fig. 5D). In
contrast, wild type K562 cells did not adhere at all.

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Fig. 5.
Cell attachment to gC1q-1 is
4 integrin-dependent.
A, attachment of SW982 cells in the presence of different
function-blocking mAbs. The miniplate wells were coated with 10 µg/ml
gC1q-1. Antibodies were added at 5 µg/ml just before cell plating:
4B4 (anti- 1), TS2/7 (anti- 1), 12171 (anti- 2), F4 (anti- 3), P1H4
(anti- 4), JBS5 (anti- 5 1),
B2121 (anti- 3), and LM609
(anti- v 3). B, attachment of
Jurkat cells to gC1q-1 (circles) and to FN
(squares) coated at 10 µg/ml was performed in the presence
of varying concentrations of the function-blocking mAb P1H4
(closed symbols) and a non-function-blocking mAb (open
symbols). C, attachment of Jurkat cells in the presence
of soluble integrins. The miniplate wells were coated with 10 µg/ml
EMILIN-1 or FN. Integrins were added at 5 µg/ml just before cell
plating. Inset, protein-protein interaction in a solid phase
assay. Wells were coated with 10 µg/ml purified integrins; soluble
EMILIN-1 or FN was added at 10 µg/ml for 60 min in the presence or in
the absence of function-blocking mAbs. The wells were then incubated
with specific primary antibodies followed by horseradish
peroxidase-conjugated secondary antibodies. D, attachment of
K562 and 4/K562 cells in the presence of 10 ng/ml
12-O-tetradecanoylphorbol-13-acetate. The miniplate wells
were coated with 10 µg/ml gC1q-1. Antibodies were added at 5 µg/ml
just before cell plating: 4B4 (anti- 1) and P1H4
(anti- 4). Results shown are the average ± S.D. of
triplicate experiments.
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SW982 Cells Do Not Form Focal Adhesion Plaques or Stress Fibers
after Attachment to EMILIN-1 and gC1q-1--
To distinguish between
cell tethering to and cell spreading on the substratum, the cell
morphology of adherent cells was examined by confocal microscopy and
double immunofluorescence staining with phalloidin and anti-paxillin
antibodies. In accord with the different strengths of cell adhesion,
there was a pronounced qualitative difference in the morphology of
cells attached to EMILIN-1 compared with the cells plated on FN or VN.
As expected, cells attached to FN and VN appeared well spread out on
the substrate, whereas cells attached to EMILIN-1 were much smaller and
displayed wide ruffles, extending in multiple direction (Fig.
6A). Phalloidin staining of
cells spread on FN and VN revealed a high number of actin-containing
stress fibers. In addition, paxillin localized to large focal contacts
at the tips of stress fibers. In contrast, phalloidin staining of cells
attached to EMILIN-1 revealed that the actin organization was mainly
along the cell periphery at the level of extended cell protrusions,
reflecting a different organization status of the actin cytoskeleton
compared with the cells on FN or VN; paxillin was distributed evenly in
the cell cytoplasm without any apparent focal contact formation.

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Fig. 6.
SW982 cell attached onto EMILIN-1 and
gC1q-1 do not form stress fibers or focal adhesions. A,
cells were plated for 30 min on substrate-coated coverslips, fixed, and
stained with Texas Red phalloidin and anti-paxillin antibody followed
by fluorescein isothiocyanate-conjugated rabbit anti-mouse IgG. The
overlays clearly demonstrate stress fibers and focal adhesion formation
only on cells attached to FN and VN. B, cells were plated on
CS-1, gC1q-1, and C1q-C' and stained as in A.
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Because SW982 cells express several integrins that can recognize FN or
VN, whereas cell adhesion to EMILIN-1 depends only upon
4
1, the morphology of cells attached to
gC1q-1 and CS-1 was then compared. Under these conditions, the
4
1-dependent adhesion to CS-1
was different compared with adhesion to FN, with the cells displaying a
smaller size and lacking clearly visible phalloidin-positive stress
fibers and focal contacts (Fig. 6B). In analogy with the
pattern detected on EMILIN-1, adhesion to gC1q-1 was accompanied by the
formation of several cellular projections positive for colocalized
actin and paxillin, but again with no detectable stress fibers nor
focal contacts. Cells attached to C1q-C', shown for comparison, also
were poorly spread with no visible stress fibers or focal contacts.
4
1 is not localized in focal adhesions in
most cell types, and it has been reported that the
4
cytoplasmic tail confers a migratory activity (37) and that it promotes
broad lamellipodia protrusions (38). Because the pattern of cell
adhesion to EMILIN-1 and/or gC1q-1 was suggestive of a promigratory
function, cells were added to the upper side of Transwell inserts. As
seen in Fig. 7, cell migration
extensively toward the migration was fully abrogated by the addition of
anti-
1 or anti-
4 function-blocking
mAbs.

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Fig. 7.
Cell migration assay. The underside of
the insert membrane was coated with 20 µg/ml EMILIN-1 or gC1q-1 at
4 °C in bicarbonate buffer and blocked with 1% (w/v) BSA for 1 h at room temperature. HT-1080 cells (105) were aliquoted
to the upper side in the presence or in the absence of blocking mAbs.
The migratory ability of HT-1080 cells was evaluated as the number of
cells migrated/field after 4 h.
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DISCUSSION |
In this study we have demonstrated that the gC1q-1 domain of
EMILIN-1 is recognized by the integrin
4
1. In fact, cell adhesion and receptor
binding were inhibited by function-blocking anti-
1 and
anti-
4 mAbs. This is the first description of
4
1 recognition of a gC1q domain and
indicates that EMILIN-1 appears to be distinct from several of the
elastic fiber-associated constituents that use mainly
v
3 but also
v
1,
v
5,
3
1, or
9
1
for cell adhesion and spreading (18-22). Recognition of the gC1q-1
domain did not require exogenous activation of
4
1 by the addition of Mn2+ or
anti-
1-activating mAbs, but the presence of
Ca2+ or Mg2+ was necessary because EDTA
treatment fully abolished cell adhesion.
The finding that gC1q-1 maintains full capability of cell attachment,
although at a higher molar ratio compared with EMILIN-1, suggests that
the rest of the molecule does not impose conformation constraints at
least to affect its adhesive function. The differential activity in
molar terms between EMILIN-1 and gC1q-1 is not without precedent for
other ECM adhesive proteins. For example, the central cell binding
domain of FN showed a 100-200-fold reduced adhesion activity compared
with intact FN (39), and the recombinant globular domain 3 of LN-5 was
about 250-fold less effective than LN-5 in molar terms (40).
However, in those cases synergistic cell binding sites contributing to
cell adhesion are present (41, 42) or hypothesized (40).
EMILIN-1-dependent cell adhesion was apparently fully
accounted for by the gC1q-1 domain because a mAb against gC1q-1 could
totally abrogate cell adhesion to EMILIN-1, and the integrin
1 subunit-blocking mAb could block cell adhesion to gC1q-1 as well as EMILIN-1. Thus, the trimeric nature of gC1q-1 and the
packing geometry of these globular domains represent a sufficient
structural organization to promote cell attachment even at moderately
low ligand densities. The few previous studies on cell adhesion using
gC1q domain-containing substrates such as type VIII collagen have
indicated that integrins of the
2
1 type
were involved (43, 44); however, because in both reports the
pepsin-resistant collagenous part of the molecule devoid of the
globular C1q domain was used, the cell adhesive function was not
attributable to the gC1q domain.
C1q-C', the prototype of the C1q/tumor necrosis factor superfamily, is
a promiscuous protein, and its function is not limited to the
recognition and triggering of the classical complement pathway, but
following interaction with cells it can directly mediate several immune
effector functions such as phagocytosis, chemotaxis, and the generation
of procoagulant activity (45). C1q-C' binds via either of its two
structurally and functionally different domains to a large number of
proteins among which cell-associated receptors/binding proteins with
quite diverse molecular structure and function were identified on
several cell types (35, 46). Although the significance of those
cell-associated molecules has been undermined by the controversy
surrounding each of the identified molecules, among the various
receptors/binding proteins gC1q-R, a predominantly mitochondrial
protein (47) that can also be expressed on the cell surface, was shown
to interact with the globular C-terminal domain of C1q-C' (48) and
promote cell adhesion (35). A recent study supports the hypothesis that
C1q-C'-mediated endothelial cell adhesion and spreading require
cooperation between gC1q-Rs and
1 integrins (34). This
conclusion was based on the finding that anti-
1 4B4
blocked both cell adhesion and spreading and that anti-
5
mAb blocked spreading. In addition, cell spreading on and in part cell
adhesion to C1q-C' were also inhibited by RGDS-containing peptides
(34). The present data on C1q-C' are apparently not in accord with
those of Feng et al. (34), but in that study cell adhesion
to and spreading on C1q-C' were evaluated at 1-2 and 6-7 h,
respectively, and not at 30 min as in the present study. For this
reason the reported involvement of integrins might well have been a
secondary phenomenon. Furthermore, that cell attachment to EMILIN-1 and
gC1q-1 was fully dependent upon a
1 integrin,
4
1, whereas cell attachment to C1q-C' was
unaffected by a
1-blocking mAb, highlights the presence
of different cell adhesion mechanisms to account for interaction with
gC1q domains.
4
1 integrin mediates tethering, rolling,
and firm arrest on VCAM-1, which is expressed on endothelial cells at
sites of inflammation (49).
4
1 also binds
to alternatively spliced variants of FN (50) which contain connecting
segment CS-1. The
4
1 integrin shows a
broader ligand binding specificity than most other members of the
integrin family (51) because overlapping but distinct binding
mechanisms exist for different ligands, and distinct conformational
changes are induced upon engagement by different ligands (52). In fact,
several additional naturally occurring ligands for
4
1 have been reported recently (53-59), and there are no sequence homologous to the known
4
1 recognition motifs QIDSP (VCAM-1),
EILDV (FN), MLDG (EC3 disintegrin peptide) within the gC1q-1 domain of
EMILIN-1 (1).
Because
4
1 exhibits a predominantly
leukocyte expression pattern, the finding that nonhematopoietic cells
could attach very efficiently to gC1q-1 via
4
1 was not expected. The present finding that both hematopoietic and nonhematopoietic cells attach to gC1q-1 via
4
1 without any prior artificial cellular
activation or immunological manipulation of the integrin receptor
complex suggests that, irrespective of the cell-specific constraints,
the constitutive activation status of
4
1
is sufficient to determine cell attachment to gC1q-1. Furthermore, and
in accord with the literature (60), only
4/K562 needed
cellular activation to display cell adhesion. Melanoma cells were
reported to bind to CS-1 peptide according to
4
1 density (61). In addition, in the
developing human aorta
4
1 was detected on
smooth muscle cells at 10 weeks, but its expression was reduced within
the 24th week of gestation and disappeared in the adult aortic media
(62). However, smooth muscle cells from intimal atherosclerotic
thickening of adult aorta reexpress
4
1
(62), suggesting a possible role in the induction of smooth muscle
differentiation. In accord with this notion is the finding that
4
1 activates the L-type calcium channels
in vascular smooth muscle and causes arteriole vasoconstriction,
pointing to an involvement in the modulation of vascular tone and in
vascular responses to mechanical signals, such as pressure and flow
(63). The elevated expression of EMILIN-1 detected in vascular tissues
(3) is in keeping with the above results and hypothesizes a role also for EMILIN-1 in development as well as pathological processes of large vessels.
Cell spreading is a complicated phenomenon that requires active
remodeling of adhesion sites to enable cells to extend processes subsequent to attachment. A current model for cell adhesion is that
there is a hierarchical mechanism for the formation of focal adhesions
in which paxillin accumulation and a small cluster of ligand-bound
integrins favor the nucleation of additional signaling and structural
molecules joining the complex (64). After the integrin-substrate
interaction, cells increase their surface contact area with the ECM
through formation of actin microfilaments and initial cell spreading.
This attachment stage is considered an intermediate stage between that
of the weak initial contact and the strong adhesion to the appropriate
ECM ligands. Most ECM constituents including FN and VN promote cell
adhesion and cause cytoskeletal reorganization as described above.
However,
4
1-dependent
interactions that were studied extensively in hematopoietic cells have
shown that the initial and intermediate stages of cell adhesion,
i.e. attachment and spreading, were supported, whereas focal
adhesion and stress fiber formation, characteristic of strong cell
adhesion, are rarely if ever observed (65). The distribution pattern of actin and paxillin suggests that attachment to EMILIN-1 and gC1q-1 leads to an accumulation of ruffles-inducing signals; this would explain the lack of polarization and of stress fiber formation. In an
elegant study using
4/green fluorescent fusion proteins to evaluate the role of
4 in lamellipodia protrusions in
response to scratch-wounding, Yang and collaborators (38) demonstrated that
4
1 forms transient puncta that do
not colocalize with paxillin-positive focal adhesion complexes. It has
been suggested that intermediate states of adhesion favor cell motility
and that cell migration is diminished in cells exhibiting strong
adhesion (66). Thus, whereas
5
1 in focal
complexes mediates cell substratum adhesion stabilizing it (67),
4
1 promotes lamellipodia formation
independent of focal adhesion complexes (38). Accordingly,
4
1-dependent migration toward
gC1q-1 was demonstrated in the present study. The formation of focal
adhesion stabilized by stress fibers is disadvantageous for cell
detachment, and the absence of mature focal adhesions has long been
associated with a motile phenotype. The lack of stress fibers and focal
adhesions in cells attached to EMILIN-1 and gC1q-1 indicates that
cells, by binding via
4
1 to these
ligands, are preferentially stimulated to migrate rather than to adhere firmly.