Download Cell-type Specific Recognition of RGD- and Non

THE JOURNAL OF BIOLOGICAL CHEMISTRY
© 1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 271, No. 9, Issue of March 1, pp. 4916 –4922, 1996
Printed in U.S.A.
Cell-type Specific Recognition of RGD- and Non-RGD-containing
Cell Binding Domains in Fibrillin-1*
(Received for publication, September 21, 1995, and in revised form, November 24, 1995)
Hiroshi Sakamoto‡, Thomas Broekelmann§, David A. Cheresh¶, Francesco Ramirezi,
Joel Rosenbloom**, and Robert P. Mecham‡§‡‡
Departments of ‡Cell Biology and Physiology and §Medicine, Washington University School of Medicine,
St. Louis, Missouri 63110, ¶Department of Immunology, Scripps Research Institute, La Jolla, California 92037,
iBrookdale Center for Molecular Biology, Mt. Sinai School of Medicine, New York, New York 10029, and
**School of Dental Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104
The fibrillins are large glycoprotein components of
10-nm microfibrils found in the extracellular matrix of
most tissues. Microfibrils play a role in elastic fiber assembly and serve to link cells to elastic fibers in the
extracellular matrix. To determine whether fibrillin-1
specifically interacts with receptors on cells from fibrillin-rich tissues, we evaluated whether two cell types
that produce different types of fibrillin can adhere to
purified fibrillin-1 in cell adhesion assays. Our results
indicate that both cell types attach and spread on fibrillin-1 and that the RGD sequence in the fourth 8-cysteine
motif mediates this interaction. Fibroblast attachment
to fibrillin-1 was sensitive to inhibition by antibodies to
the avb3 receptor and by peptides encoding the RGD
sequence in fibrillin-1 and the second RGD sequence in
fibrillin-2. In contrast, adhesion of auricular chondroblasts to fibrillin-1 was only partially inhibited by these
reagents, suggesting that some cell types recognize a
second, non-RGD binding site within the fibrillin molecule. These findings confirm and extend ultrastructural
studies that suggest a direct interaction between microfibrils and the cell surface and provide a functional
explanation for how this association occurs.
Microfibrils are filamentous structures with a diameter of
10 –12 nm found in the extracellular matrix of most tissues.
Although usually associated with elastic fibers, microfibrils
devoid of elastin are also found in abundance in tissues that are
subject to mechanical stress where they are thought to provide
a link between the cell and underlying elastic structures (1).
The term microfibril was first used by Low (2) as an arbitrary
morphological descriptive term for all filaments in the extracellular space with a diameter of less than 20 nm lacking a
typical collagen banding pattern. Recent studies have proven
this definition to be too broad since some filaments that fit
Low’s description are, in fact, collagenous in nature or react
with antibodies to known matrix molecules such as fibronectin
or proteoglycans. It is thus apparent that many matrix components may exist in the form of microfibrils and that morphology
* This work was supported by National Institutes of Health Grants
HL-26499 and HL-41926 (to R. P. M.), AR-41474 (to J. R.), CA50286
and CA45726 (to D. A. C.), and AR42044 (to F. R.). This is article 165
from the Brookdale Center for Molecular Biology. The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
‡‡ To whom correspondence should be addressed: Dept. of Cell Biology and Physiology, Washington University School of Medicine, Box
8228, 660 South Euclid Ave., St. Louis, MO 63110. Tel.: 314-362-2254;
Fax: 314-362-2252.
alone cannot be used to characterize these structures (3).
Within the past few years, the direct biochemical and immunological characterization of microfibrillar components has led
to a more accurate definition of a microfibril. Several proteins,
including AMP, microfibril-associated glycoprotein (MAGP),1
fibrillin-1, and fibrillin-2, have been shown to be major components of these structures (4 –7), although other proteins are
associated with microfibrils in some tissues (4, 8). While our
understanding of the composition of these unique filaments is
still emerging, immunological studies suggest that fibrillin is a
common constituent of all microfibrils, and hence, the presence
of this protein provides a minimal definition of what can be
called a microfibril. Even so, this definition includes an element
of complexity since differences in the spatial and temporal
expression of fibrillin-1 and fibrillin-2 (6, 9) implies that microfibrils may consist of one protein or the other, or both.
When associated with elastic fibers, microfibrils are thought
to provide the basic scaffolding for elastic fiber organization.
The initial stages of assembly occur at regions of the cell surface where microfibrils frequently appear attached to the
plasma membrane. Ultrastructurally, these areas of attachment resemble focal contacts with an abundance of microfilaments and cytoskeletal elements on the cytoplasmic side of the
membrane (10). An association with membrane-associated
dense plaques has also been observed for microfibrils that serve
to anchor endothelial and epithelial cells to underlying elastic
fibers. The propensity of cytoskeletal elements to cluster where
microfibrils contact the cell surface suggest that protein components of the microfibril interact with cell-surface receptors
which, in turn, serve as a transmembrane link between microfibrils and components of the cytoskeleton.
Of the known microfibrillar proteins, the fibrillins are the
best candidates for interacting with cell surface receptors. The
two known fibrillin gene products manifest the same general
motif structure, but functional differences between the proteins
are suggested by a disparity in their pattern of expression (6) as
well as by the distinct clinical phenotypes resulting from mutations in one or the other genes (11). Structural similarities in
fibrillin-1 and fibrillin-2 consist of clusters of epidermal growth
factor-like domains separated by 8-cysteine domains that were
first described in transforming growth factor-b1-binding protein (11, 12). Overall, the amino acid homology between the two
fibrillins approaches 70%, and all but one of the putative Nlinked glycosylation sites are conserved. The most striking
1
The abbreviations used are: MAGP, microfibril-associated glycoprotein; FCL, fetal bovine ligamentum nuchae; FBC, fetal bovine auricular
cartilage; LTBP-2, latent transforming growth factor-b-binding protein-2; DMEM, Dulbecco’s modified Eagle’s medium; BSA, bovine serum albumin.
4916
Cell Receptor for Fibrillin
FIG. 1. Domain map of fibrillin-1 and fibrillin-2. Letters at the
top indicate the five structurally distinct regions of the molecule. The
repeating structural elements are defined at the bottom of the figure.
The location of RGD sequences are shown along with the synthetic
peptides that were used for adhesion assays and antibody production.
sequence divergence is found at the amino terminus (Fig. 1,
domain A, 19% homology) and at the end of the first 8-cysteine
motif where a proline rich sequence in fibrillin-1 is replaced
with a glycine rich sequence in fibrillin-2.
The possibility that fibrillin might interact with cell receptors is suggested by the presence of RGD sequences in both
fibrillin molecules. Fibrillin-1 contains one RGD sequence located in the fourth 8 cysteine. Fibrillin-2, in contrast, has two
RGD sequences: one in the same position as fibrillin-1, and a
second in the third 8-cysteine motif (see Fig. 1). This second
RGD sequence is surrounded by hydrophobic amino acids
whereas the RGD in the fourth 8-cysteine motif of fibrillin-1
and -2 is surrounded by polar and charged residues. At all three
sites, the RGD tripeptide is located in the middle of a 13–20amino acid sequence that is flanked on both ends by cysteine
residues. Disulfide bonding of these cysteines would produce a
finger-like loop structure with the RGD sequence near the end
of the loop.
In this study we evaluated whether fibrillin specifically interacts with receptors or binding proteins on cells found in
fibrillin-rich tissues. Using adhesion assays, we have shown
that cells specifically interact with fibrillin-1 and that the
RGD-containing domain defines a major cell binding epitope.
The integrin avb3 is the adhesion receptor responsible for
binding, although other integrins may interact with this sequence under some conditions. Our data also suggest a second,
non-RGD receptor-binding site in fibrillin, that is recognized by
some cell types.
EXPERIMENTAL PROCEDURES
Materials—Ellman’s reagent, Immunopure Sulfo-NHS-Biotinylation
kit, IODOGEN, and m-maleimidobenzoyl-N-hydroxysuccinimide ester
were purchased from Pierce. Plates used for adhesion assays were from
Costar. Antibodies to fibrillin-1 and fibrillin-2 were raised against synthetic peptides (see below) encoding the RGD sequences of these proteins (9). Rabbit polyclonal antibodies to a1, a3, a5, and b1 integrin
subunits, and a rat monoclonal to the b3 subunit were generously
provided by Dr. John McDonald. Polyclonal antibodies to fibronectin,
vitronectin, and to the a5b1 integrin were from Telios Pharmaceuticals.
Antibodies to av (LM142) and avb3 (LM609) have been described previously (13). All other reagents were from Sigma.
Synthetic Peptides and Peptide Antibodies—Peptides to the RGD
sequences in human fibrillin-1 and fibrillin-2 were synthesized using an
ABI model 431A synthesizer employing t-butoxycarbonyl chemistry.
The sequence of each peptide was confirmed by sequence analysis using
an ABI 473 protein sequencer. Peptide Fib-1d-RGD (YLDIRPRGDNGDTA) includes the RGD sequence of fibrillin-1, peptide Fib-2-RGD1
(AGFANRGDVLTG) contains the first RGD sequence of fibrillin-2 located in the third 8-cysteine domain, and peptide Fib-2-RGD2 (YLKFGPRGDGSL) contains the second RGD sequence in fibrillin-2. The
locations of RGD sequences in fibrillin-1 and fibrillin-2 are shown
schematically in Fig. 1. Control peptides included Fib-1d-RGQ (Gln
4917
substituted for Asp, YLDIRPRGQNGDTA) and GRGDSP. Antibodies to
the fibrillin peptides have been described previously (9). By immunoblot
analysis, the Fib-1 peptide antibody did not react with fibrillin-2 or
fibronectin. Conversely, the antibody to the Fib-2 peptide reacted with
fibrillin-2 but not fibrillin-1. Antibodies to fibronectin did not react with
either fibrillin-1 or fibrillin-2.
Fab fragments of the Fib-1d-RGD antibody were generated by pepsin
digestion and reductive alkylation. IgG purified from the immune serum was dialyzed into 70 mM sodium acetate, 50 mM NaCl, pH 4.0,
concentrated to 5 mg/ml, and treated with pepsin overnight at 37 °C at
a 1:33 enzyme to substrate ratio. Following buffer exchange into 0.5 M
Tris, pH 8.3, the digest was reduced with 10 mM 2-mercaptoethanol for
1 h at room temperature. Iodoacetamide was added to 12 mM, and the
digest was incubated for 15 min on ice in the dark. The Fab fragments
were then dialyzed into 7.5 mM Tris, 150 mM NaCl, pH 7.5, and purified
on a 1.5 3 90-cm Sephacryl S-200 column equilibrated in the same
buffer.
Cells—Fibroblasts from fetal bovine ligamentum nuchae (FCL cells)
and chondroblasts from fetal bovine auricular cartilage (FBC cells)
were cultured as described elsewhere (14). Cells were maintained in
Dulbecco’s modified Eagle’s medium (DMEM) containing 10% calf serum and were used before passage 3.
Fibrillin Purification and Radiolabeling—Microfibrillar proteins
were isolated from fetal bovine ligamentum nuchae using a modification of the reductive saline procedure of Gibson et al. (4). Proteinase
inhibitors e-amino caproic acid (10 mM), EDTA (5 mM), benzamidine (5
mM), phenylmethylsulfonyl fluoride (1 mM), sodium azide 0.1% (w/v),
and N-ethylmaleimide (2 mM) were included in all buffers and all
extraction steps were done at 4 °C. Freshly harvested tissue (50 g) was
homogenized and extracted twice for 1 h with 250 ml of 20 mM HEPES,
pH 7.4, 150 mM NaCl, 0.1% Nonidet P-40 followed by two overnight
extractions with the same volume of 1 M NaCl, 20 mM HEPES buffer,
pH 7.4. The tissue pellet was then extracted four times over 48 h with
250 ml of 6 M guanidine HCl, 50 mM HEPES buffer, pH 8.0, and washed
with HEPES, pH 7.4, 150 mM NaCl until the A280 of the supernatant
was less than 0.005. The insoluble residue was finally extracted twice
over 24 h with 200 ml HEPES, pH 7.4, containing 25 mM dithiothreitol.
The dithiothreitol extracts were combined, clarified by centrifugation,
and fractionated on a Sepharose CL-4B column (1 cm 3 1.5 m) using 50
mM HEPES, pH 7.4, 4 M guanidine HCl as the buffer. Column fractions
were monitored for fibrillin-1, fibrillin-2, latent transforming growth
factor-b-binding protein-2 (LTBP-2), and fibronectin by immunoblot
analysis. Fibrillin-containing fractions were pooled, dialyzed against
HEPES-urea buffer (6 M urea, 20 mM HEPES, pH 7.4), and fractionated
on a DEAE-Sepharose column (1.5 3 10 cm) equilibrated in HEPESurea buffer and developed with a linear 0 to 0.3 M NaCl gradient. Final
purification was achieved using a heparin-Sepharose column (1 ml
volume) developed with the same buffer system as described for DEAE.
The concentration of purified fibrillin was determined by amino acid
analysis after alkylation as described by Gibson et al. (4).
Fibrillin was radiolabeled by mixing approximately 30 mg of purified
protein with 300 mCi of Na125I in IODOGEN-coated glass tubes for 10
min at room temperature. Labeled protein was separated from free
iodine by gel filtration using Sephadex G25.
Cell Attachment Assay—Polystyrene 96-well plates were incubated
at room temperature overnight with fibrillin-1 diluted in Tris-buffered
saline or bovine serum albumin (BSA) conjugated with synthetic peptides in the same buffer. Unbound sites were then blocked with 1 mg/ml
BSA in phosphate-buffered saline. Using 125I-labeled fibrillin-1 to evaluate coating efficiency, we found that approximately 90% of the added
protein up to a concentration of 30 mg/well bound to the plate. The
optimal concentration of fibrillin that supported cell adhesion was determined by coating wells with dilutions of fibrillin ranging from 0 –200
mg/well. First to third passage cells were harvested by trypsinization,
washed once in complete DMEM supplemented with 10% calf serum
and twice in serum-free DMEM containing 1 mg/ml BSA. Cells in
200-ml aliquots of binding buffer (DMEM containing 1 mg/ml BSA) were
added to each well and incubated at 37 °C for periods of up to 2 h.
Nonadherent cells were removed by gentle rinsing, and the number of
adherent cells was quantified using the hexosaminidase assay of Landegren (15). Maximal cell attachment was achieved at concentrations of
10 ng of fibrillin-1/well, which was the concentration used for all adhesion studies.
Peptide competitors of cell adhesion were added to the wells at the
same time as the cells. In experiments using antibodies, the cell suspension and the fibrillin-containing well were preincubated for 30 min
at room temperature with antibodies dissolved in binding buffer. Adhesion was then carried out in the usual manner.
4918
Cell Receptor for Fibrillin
Immunofluorescence Analysis of Receptor Localization—Adherent
cells were suspended by a 10-min treatment with Versene, washed, and
allowed to adhere to multichamber slides coated with fibrillin-1 in the
absence of serum for 1 h at 37 °C. The nonadherent cells were then
removed, and the adherent cells were fixed with 3% paraformaldehyde
in Hanks’ balanced salt solution for 30 min. The fixed cells were permeabilized with 0.5% Triton X-100 for 1 min, washed, and blocked with
5% BSA in phosphate-buffered saline. The permeabilized cells were
stained with antibody to the b3 integrin subunit for 60 min at room
temperature, washed, and incubated with fluorescein isothiocyanateconjugated goat anti-rat IgG. After washing, the slides were analyzed
by fluorescence microscopy.
RESULTS
Purification of Bovine Fibrillin-1—Microfibrillar proteins
were extracted from fetal bovine ligamentum nuchae using a
modification of the reductive saline procedure of Gibson et al.
(4). This approach utilizes repeated extraction with high salt
and guanidine to remove soluble proteins before microfibrillar
components are solubilized with a final extraction incorporating reducing agent. The ligamentum nuchae was chosen for
this study because it is highly enriched in microfibrils containing fibrillin-1 with only low levels of fibrillin-2 (9). Throughout
the isolation procedure, extraction supernatants and column
fractions were monitored by immunoblot analysis for the presence of fibrillin-1, fibrillin-2, MAGP, fibronectin, vitronectin,
and LTBP-2. This latter protein, which has many properties in
common with fibrillin (16), has been shown to be an integral
component of microfibrils and a potential contaminate of fibrillin preparations (17).
Dot blot immunoassay of sequential extraction supernatants
showed that fibronectin was present at all stages of the purification, although only small amounts of the protein were detected in the final extract. Vitronectin, in contrast, was found
in the first pellet wash and was not found in later extractions.
Gibson et al. (4) have shown that the final reductive extract is
highly enriched in microfibrillar components and contains essentially five proteins: fibrillin, LTBP-2, MP78/70, MAGP, and
MP25. When this extract was fractionated over a CL-4B column, fibrillin-1 was found to elute as a broad peak shortly after
the void volume. On SDS-polyacrylamide gel electrophoresis,
the leading fractions in this peak were shown to contain a
single protein at ;340 kDa that reacted with the Fib-1 antibody. This protein did not react with antibodies specific for
fibrillin-2, fibronectin, or vitronectin, nor were fibronectin or
vitronectin detected in these fractions by Western blot (not
shown). Confirmation that the 340-kDa protein was fibrillin-1
was obtained by partial sequence analysis of a cyanogen bromide peptide generated from the parent protein (data not
shown). Later fractions in this peak contained several lower
molecular weight bands that reacted with the Fib-1 antibody
and were presumed to be degradation products of fibrillin-1.
Fibronectin and LTBP-2 eluted in fractions that overlapped
with the receding end of the fibrillin-1 peak (Table I). Smaller
microfibrillar components, including MP78/70 and MAGP (4),
eluted later in the column run.
To increase our yield of purified fibrillin, fibrillin-1-containing fractions from the CL-4B column were pooled as indicated
in Table I and were fractionated using DEAE followed by
heparin-Sepharose. Immunoblot analysis showed that DEAE
effectively separated LTBP-2 from fibrillin-1 and a small
amount of fibronectin, but the later two proteins eluted as
overlapping peaks which could be separated using heparinSepharose. Fig. 2 is a SDS-polyacrylamide gel electrophoresis
analysis of purified fibrillin-1 showing a single band at the
expected molecular mass of approximately 340 kDa. Western
blot analysis confirmed the absence of fibronectin in the final
product.
Cell Attachment to Fibrillin-1—To study possible interac-
TABLE I
Elution of fibrillin-1, fibronectin, and LTBP-2 from CL-4B, DEAE,
and heparin-Sepharose columns
Proteins were detected by immunoblot analysis and were subjectively
scored to indicate approximate concentrations. Relative values are not
comparable between colmns.
Fraction no.
36
CL-4B column
Fibrillin-1
Fibronectin
LTBP-2
38
40
42
44
46
48
50
52
1 1111 111 11 11 1 1 1/2
1/2 1/2 1 1 1
1 1/2
1 1 11 11 1
Pooled
30 33
DEAE column
Fibrillin-1
Fibronectin
LTBP-2
36
39 41
43
46
49
52
55
1 1111 111 1 1/2
1/2 1 1 1/2
1 111 1
Pooled
27
Heparin-Sepharose
column
Fibrillin-1
Fibronectin
LTBP-2
29
31
33
35
37
39
41
1
11
111
11
1/2
1
1
1
1
1/2
Pooled
FIG. 2. Characterization of purified fibrillin-1. Lane A shows
Coomassie Blue staining of a 5% SDS-polyacrylamide gel containing
fibrillin-1 purified from fetal bovine ligamentum nuchae (pooled fractions 29 –33 from heparin-Sepharose chromatography, see Table I).
Lanes B and C are immunoblots transferred from 10% gels developed
with an antibody to fibronectin and lanes D and E are developed with
the fibrillin-1d antibody. Lanes B and D contain a commercial preparation of fibronectin and lanes C and E contain the fibrillin preparation
shown in lane A. Molecular weight standards are 3 103.
tions between fibrillin and elastin-producing cells, freshly harvested FCL and FBC cells were tested in a cell adhesion assay
for attachment to fibrillin-1, or to the control protein, BSA. Fig.
3 shows that both cell types attached to fibrillin-1 but failed to
adhere to BSA. The number of cells showing positive adherence
in each assay was proportional to the amount of protein ligand
used to coat the microtiter well. Thus, for both cell types,
attachment appeared to be directly to fibrillin-1. Maximal cell
attachment was achieved at concentrations of 10 ng fibrillin-1/
well. Both cell types readily spread on the fibrillin matrix.
Cell Receptor for Fibrillin
4919
FIG. 3. Adhesion of FBC and FCL cells to fibrillin-1. Wells were
incubated with fibrillin-1 solutions ranging in concentration from 0.01
to 200 ng/well. Chondroblasts (FBC cells, squares) and fibroblasts (FCL
cells, circles) were allowed to adhere in the wells for 2 h. After gentle
washing, adherent cells were quantified using a colorometric determination (405 nm) of hexosaminidase. Open symbols indicate adhesion to
fibrillin-1. Filled symbols indicated adhesion to wells coated with 100
mg/well BSA. Mean 6 standard deviation of triplicate determinations.
Inhibition of Cell Attachment by RGD-containing Peptides
from Fibrillin-1—Specific cell binding sites within the fibrillin
molecules have been suggested by the presence of RGD sequences (see Fig. 1). To determine whether these sites mediate
attachment and spreading of FCL and FBC cells, synthetic
peptides encoding these sequences were added together with
cells to fibrillin-1-coated wells. As shown in Fig. 4A, the number of FBC cells showing attachment to fibrillin-1 was reduced
in a concentration-dependent fashion by the Fib-1d-RGD and
GRGDSP peptides. However, maximal inhibition by Fib-1dRGD peptide was never greater than 50%, even at extremely
high peptide concentrations. Synthetic GRGDSP peptide was a
slightly better inhibitor than the fibrillin-1 sequence but even
at the highest concentration tested, GRGDSP was only able to
inhibit binding by ;65%. Attachment of FCL cells, in contrast,
was completely inhibited by GRGDSP and was inhibited ;70%
by Fib-1d-RGD peptide (Fig. 4B). Peptide Fib-2-RGD2, encoding the RGD sequence in fibrillin-2 located in the same 8-cysteine domain as fibrillin-1, also inhibited adhesion of FBC cells
to purified fibrillin-1, although to a lesser extent (Fig. 5). Peptide Fib-2-RGD1 was ineffective at blocking adhesion of either
cell type, as was the control peptide, Fib-1d-RGQ. It is important to note that FBC cells that remained attached to fibrillin-1
in the presence of Fib-1d-RGD and GRGDSP peptides had a
rounded morphology and did not spread during the 2-h assay
period (Fig. 6).
The ability of the fibrillin-1 RGD domain to support adhesion
was tested directly by adding Fab fragments of affinity purified
Fib-1d-RGD antibody to the adhesion assay. Fig. 7 shows that
FCL cell attachment to fibrillin-1 was inhibited by 70% in the
presence of 100 mg/ml of this antibody. FAB fragments of an
antibody to another microfibrillar component, MAGP, had no
effect on adhesion of FCL cells and served as a negative control.
Antibodies Directed to avb3 Integrin Block FCL and FBC
Adhesion to Fibrillin-1—Because affinity chromatography and
peptide blocking studies were strongly suggestive that avb3 is
a major adhesion receptor for fibrillin-1, we examined whether
antibodies to the avb3 receptor complex could inhibit binding of
FCL and FBC cells to fibrillin-coated dishes. Fig. 8 shows the
results of an adhesion assay where cells were preincubated
FIG. 4. Inhibition of FBC and FCL cell attachment to fibrillin-1
by RGD-containing peptides. A, FBC cells in medium containing
increasing concentrations of Fib-1d-RGD (squares) and GRGDSP (open
circles) peptides were added to microtiter plates coated with fibrillin-1.
Peptide Fib-1d-RGQ (filled circles) served as a control. Mean 6 standard deviation of triplicate determinations. B, comparison of 100 mM
peptide inhibition of FCL and FBC cell binding to fibrillin-1. After
gentle washing, adherent cells were quantified as described in Fig. 3.
For Panel B, values are expressed relative to cell adherance in the
absence of added peptide.
with an antibody to the avb3 complex. This antibody was able
to block binding of FBC cells by ;60%, which was similar to the
inhibition of attachment observed with the Fib-1d-RGD and
GRGDSP peptides. An antibody directed against the av subunit alone did not prevent cell attachment and was used as a
negative control. Interestingly, an antibody to the fibronectin
receptor (a5b1) stimulated cell adhesion (Fig. 8).
Integrin avb3 Localizes to Focal Contacts in Cells Attached to
Fibrillin-1—As shown in Fig. 9, FCL cells which spread on
purified fibrillin-1 express b3 in focal contacts, indicating that
avb3 expression promotes the assembly of adhesion plaques
and organization of the cytoskeleton leading to cell spreading.
The b3 staining pattern of cells plated on fibrillin is identical to
the distribution of focal contacts formed when these cells attach
to plates coated with serum.
DISCUSSION
The objective of this study was to determine whether fibrillin-1 could specifically interact with binding proteins on the
4920
Cell Receptor for Fibrillin
FIG. 5. Effects of fibrillin-2 sequences on cell attachment. FBC cells
in medium containing 100 mM Fib1d-RGD, Fib-2-RGD1, Fib-2-RGD2, or
GRGDSP peptides were added to microtiter plates coated with fibrillin-1. After
gentle washing, adherent cells were
quantified as described in Fig. 3. Values
are expressed relative to cell adherance
in the absence of added peptide. Mean
6 standard deviation of triplicate
determinations.
FIG. 7. Antibody to RGD sequence in fibrillin-1 blocks cell
adhesion. Fibrillin-containing wells were incubated with 100 mg/ml of
Fab fragments of antibody Fib-1d-RGD. FCL cells were added and the
number of cell adhering to the fibrillin was determined after 2 h of
incubation. Fab framents of an antibody to MAGP served as a negative
control. Adherent cells were quantified as described in Fig. 3.
FIG. 6. Phase-contrast micrograph of FBC cells attached to
fibrillin-1 in the presence (bottom) and absence (top) of Fib-1dRGD peptide.
surface of two phenotypically distinct cell types that reside in
fibrillin-rich tissues: (a) fibroblasts from the ligamentum nuchae (FCL cells) and (b) chondroblasts from auricular cartilage
(FBC cells). Both cell types are specialized to synthesize large
amounts of fibrillin, but differ in the fibrillin types they produce, with fibrillin-1 being the major form secreted by FCL
cells and fibrillin-2 by FBC cells (9). Thus, it was of great
interest to determine whether these two cell types demonstrate
adhesive properties that correlate with their differing fibrillin
phenotypes.
When tested in adhesion assays, we found that purified
fibrillin-1 was an adhesive protein for both cell types and that
the RGD-containing domain defined a major cell binding
epitope. The importance of the RGD domain for cell interactions with fibrillin was established using soluble peptides as
competitive inhibitors and by comparing cell attachment and
spreading on the intact, purified protein. Our results suggest
that the RGD sequence in the fourth 8-cysteine motif in both
fibrillin-1 and fibrillin-2 is active in cell attachment, and is
most likely recognized by the avb3 receptor. The RGD sequence
in the third 8-cysteine domain of fibrillin-2 may be either
inactive or specific for a different receptor. The inability of this
sequence to block adhesion to fibrillin demonstrates an element
of specificity in the interaction and suggests that inhibition is
not a general property of any RGD peptide.
As with many other matrix proteins, cell binding to fibrillin
appears to be cell-type specific. This was suggested by studies
showing that attachment of FBC cells to intact fibrillin-1 could
only be partially inhibited with RGD peptides or receptor antibodies. In contrast, adhesion of FCL cells was completely
inhibited by RGD-containing peptides. These results suggest
that FCL fibroblasts mainly recognize the RGD sequence,
whereas FBC cells recognize, in addition to the RGD sequence,
Cell Receptor for Fibrillin
FIG. 8. Effects of antibodies to avb3 and a5b1 receptors on
FBC cell attachment to fibrillin-1. FBC cells were incubated for 30
min with antibodies to avb3 (closed circles) or to a5b1 (open circles) and
added to microtiter wells coated with fibrillin-1. Squares are cells incubated with nonimmune antibody. After 2 h of incubation, adherent cells
were quantified as described in Fig. 3 and results were normalized to
cells adhesion in the absence of antibody. Mean 6 standard deviation of
triplicate determinations.
FIG. 9. Immunofluorescent detection of b3 integrin at the leading edge of a recently spread FCL cell plated on fibrillin-1coated slides. The staining pattern is characteristic of focal contacts.
The brightly staining object in the lower right is an unspread cell.
a second non-RGD site somewhere in the fibrillin-1 molecule.
The nature of the second binding site on fibrillin-1 is unknown,
although cells that attach to fibrillin in the presence of RGD
peptide fail to spread, suggesting that the receptor that binds
to this site does not interact with the cytoskeleton in a way that
promotes spreading.
An interesting and surprising result from our antibody inhibition studies was enhanced FBC cell binding to fibrillin in the
presence of an antibody to the a5b1 integrin. We interpret this
result to suggest that the antibody induces a change in either
a5b1 receptor affinity or specificity, thereby converting the
receptor to a form that can bind fibrillin. This is similar to the
finding by Faull et al. (18) that a b1 antibody stimulates adhesion of erythroleukemic cells by converting the fibronectin receptor from a low to a high affinity state. Other examples of cell
surface activation of integrin receptors have been described
elsewhere (19).
Our identification of cell binding activity in fibrillin-1 purified from tissues with reductive guanidine raises the question
4921
of whether domains responsible for cell adhesion are functional
in the native molecule or exist as cryptic sites that are activated upon protein denaturation. While the inability to extract
and purify native fibrillin-1 from normal tissues currently precludes an experimental evaluation of this question, other laboratories have obtained preliminary data suggesting that the
native molecule does specifically interact with cells. For example, Withers et al. (20) demonstrated attachment and spreading
of fibroblasts to a bacterially expressed fusion protein containing a fragment of fibrillin-1 containing the RGD sequence.
Similarly, Kielty et al. (21) found that smooth muscle cells
attach and spread on intact fibrillin-containing microfibrils
purified from fetal bovine skin. Although both studies document interactions between cells and fibrillin, neither study
identified the receptor responsible for this binding.
The biological role of receptors for fibrillin is undoubtedly
complex. Morphological studies have documented extensive interactions between cells and microfibrillar structures in the
extracellular matrix. In many instances, these microfibrils
form a continuous link between the cell and the extracellular
matrix, suggesting that microfibrils not only facilitate elastic
fiber assembly but also play a role in cell anchorage to extracellular matrix components. In skin, for example, microfibrils
anchor epidermal cells to elastic fibers in the dermis (22) and
similar “anchoring filaments” have been identified in the subendothelial matrix of lymphatic capillaries, coronary vessels,
and the aorta where they connect endothelial cells to surrounding elastic fibers (23–25). Recently, Davis (1) has shown that
these connecting filaments contain fibrillin, confirming their
identification as microfibrils. Another interesting property of
the connecting filaments is that they are anchored at the cell
surface in regions of the membrane occupied on the intracellular face by membrane-associated dense plaques. This is consistent with avb3 being a receptor for fibrillin since this integrin is known to associate into focal contacts and to organize
the cytoskeleton (26).
In addition to anchoring endothelial and epithelial cells to
underlying elastic fibers, fibrillin may also facilitate adhesion
or migration of stromal or inflammatory cells. The distribution
of fibrillin-1 and fibrillin-2 in developing tissues suggests that
both proteins are usually, but not exclusively, associated with
elastic fibers (6). In instances where there is no elastin, fibrillin
by itself or in association with other microfibrillar components
may act as a classical adhesion protein. Alternatively, at sites
of inflammation or active tissue remodeling, elastases could
strip elastin from microfibrils, thereby exposing fibrillin molecules that are otherwise masked. An invading inflammatory
cell with the appropriate receptors could then use fibrillin to
move through an elastin-rich tissue such as a blood vessel.
Although the present study focused on interactions between
fibrillin and normal, elastin-producing cells, other studies in
our laboratory have shown that fibrillin is an adhesive protein
for cells that do not make elastin, including several tumor cell
lines.2
In summary, we have found that fibrillin is capable of specifically interacting with receptors on the cell surface and that
the integrin avb3 is a receptor for both proteins. This integrin
supports cell attachment as well as spreading, suggesting a
distinct interaction with the cellular cytoskeleton. Organization of the cytoskeleton at microfibril attachment sites is critically important for elastic fiber assembly and for cell anchorage to surrounding matrix. Our findings suggest a functional
explanation for the many ultrastructural studies documenting
2
H. Sakamoto, T. Broekelmann, and R. P. Mecham, unpublished
observations.
4922
Cell Receptor for Fibrillin
the importance of interactions between cells and microfibrils in
maintaining tissue integrity.
Acknowledgments—We thank Dr. Elaine C. Davis for helpful discussion and Terese Hall, Clarina Tisdale, and Benjamin R. Mecham for
secretarial and technical assistance.
REFERENCES
1. Davis, E. C. (1994) J. Cell Sci. 107, 727–736
2. Low, F. N. (1961) Anat. Rec. 139, 250
3. Cleary, E. G., and Gibson, M. A. (1983) Int. Rev. Connect. Tissue Res. 10,
97–209
4. Gibson, M. A., Kumaratilake, J. S., and Cleary, E. G. (1989) J. Biol. Chem. 264,
4590 – 4598
5. Sakai, L. Y., Keene, D. R., Glanville, R. W., and Bachinger, H. P. (1991) J. Biol.
Chem. 266, 14763–14770
6. Zhang, H., Adfelroth, S. D., Hu, W., Davis, E. C., Sanguineti, C., Bonadio, J.,
Mecham, R. P., and Ramirez, F. (1994) J. Cell Biol. 124, 855– 863
7. Horrigan, S. K., Rich, C. B., Streeten, B. W., Li, Z. Y., and Foster, J. A. (1992)
J. Biol. Chem. 267, 10087–10095
8. Mecham, R. P., and Heuser, J. E. (1991) in Cell Biology of Extracellular Matrix,
2nd Ed. (Hay, E. D., ed), pp. 79 –109, Plenum Press, New York
9. Mariencheck, M. C., Davis, E. C., Zhang, H., Ramirez, F., Rosenbloom, J.,
Gibson, M. A., Parks, W. C., and Mecham, R. P. (1995) Connect. Tissue Res.
31, 87–97
10. Davis, E. C., and Mecham, R. P. (1993) in Tissue Engineering: Current
Perspectives (Bell, E., ed), pp. 26 –34, Birkhäuser, Boston
11. Lee, B., Godfrey, M., Vitale, E., Hori, H., Mattei, M.-G., Sarfarazi, M., Tsipouras, P., Ramirez, F., and Hollister, D. W. (1991) Nature 352, 330 –334
12. Maslen, C. L., Corson, G. M., Maddox, B. K., Glanville, R. W., and Sakai, L. Y.
(1991) Nature 352, 334 –337
13. Cheresh, D. A., and Harper, J. A. (1987) J. Biol. Chem. 262, 1434 –1437
14. Mecham, R. P. (1987) Methods Enzymol. 144D, 232–246
15. Landegren, U. (1984) J. Immunol. Methods 67, 379 –388
16. Morén, A., Olofsson, A., Stenman, G., Sahlin, P., Kanzaki, T., Claesson-Welsh,
L., ten Dijke, P., Miyazono, K., and Heldin, C.-H. (1994) J. Biol. Chem. 269,
32469 –32478
17. Gibson, M. A., Hatzinikolas, G., Davis, E., Baker, E., Sutherland, G. R., and
Mecham, R. P. (1995) Mol. Cell. Biol. 15, 6932– 6942
18. Faull, R. J., Kovach, N. L., Harlan, J. M., and Ginsberg, M. H. (1993) J. Cell
Biol. 124, 155–162
19. Hynes, R. O. (1992) Cell 69, 11–25
20. Withers, A. P., Burl, S., Faed, M. J. W., D’Arrigo, C., and Boxer, M. (1994)
J. Med. Genet. 31, 174
21. Kielty, C. M., Whittaker, S. P., Grant, M. E., and Shuttleworth, C. A. (1992)
J. Cell Sci. 103, 445– 451
22. Cotta-Pereira, G., Guerra-Rodrigo, F., and Bittencourt-Sampaio, S. (1976)
J. Invest. Dermatol. 66, 143–148
23. Leak, L. V., and Burke, J. F. (1968) J. Cell Biol. 36, 129 –149
24. Gerrity, R. G., and Cliff, W. J. (1972) Exp. Mol. Pathol. 16, 382– 402
25. Davis, E. C. (1993) Cell Tissue Res. 272, 211–219
26. Wayner, E. A., Orlando, R. A., and Cheresh, D. A. (1991) J. Cell Biol. 113,
919 –929