(Received for publication, September 21, 1995; and in revised form, November 24, 1995)
From the
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 v
3 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 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), ()fibrillin-1, and fibrillin-2, have been shown to
be major components of these
structures(4, 5, 6, 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-1-binding protein(11, 12) . Overall, the
amino acid homology between the two fibrillins approaches 70%, and all
but one of the putative N-linked glycosylation sites are
conserved. The most striking 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.
Figure 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.
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 13-20-amino 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 v
3
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.
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 90-cm Sephacryl S-200
column equilibrated in the same buffer.
Fibrillin was radiolabeled by
mixing approximately 30 µg of purified protein with 300 µCi of
NaI in IODOGEN-coated glass tubes for 10 min at room
temperature. Labeled protein was separated from free iodine by gel
filtration using Sephadex G25.
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.
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 1). 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 1and 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 heparin-Sepharose. Fig. 2is 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.
Figure 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 1). 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
10
.
Figure 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 µg/well BSA. Mean ± standard deviation of triplicate determinations.
Figure 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 ± standard deviation of triplicate determinations. B, comparison of 100 µM 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.
Figure 5: Effects of fibrillin-2 sequences on cell attachment. FBC cells in medium containing 100 µM 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 ± standard deviation of triplicate determinations.
Figure 6: Phase-contrast micrograph of FBC cells attached to fibrillin-1 in the presence (bottom) and absence (top) of Fib-1d-RGD peptide.
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. 7shows that FCL cell attachment to fibrillin-1 was inhibited by 70% in the presence of 100 µg/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.
Figure 7: Antibody to RGD sequence in fibrillin-1 blocks cell adhesion. Fibrillin-containing wells were incubated with 100 µg/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.
Figure 8:
Effects of antibodies to v
3 and
5
1 receptors on FBC cell attachment to fibrillin-1. FBC cells
were incubated for 30 min with antibodies to
v
3 (closed
circles) or to
5
1 (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. 3and results were
normalized to cells adhesion in the absence of antibody. Mean ±
standard deviation of triplicate
determinations.
Figure 9:
Immunofluorescent detection of 3
integrin at the leading edge of a recently spread FCL cell plated on
fibrillin-1-coated slides. The staining pattern is characteristic of
focal contacts. The brightly staining object in the lower right is an unspread cell.
The objective of this study was to determine whether fibrillin-1 could specifically interact with binding proteins on the 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 v
3 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, 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 5
1 integrin.
We interpret this result to suggest that the antibody induces a change
in either
5
1 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
1 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 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, 24, 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 v
3 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. ()
In
summary, we have found that fibrillin is capable of specifically
interacting with receptors on the cell surface and that the integrin
v
3 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 the importance of interactions between cells and
microfibrils in maintaining tissue integrity.