Microfibril-associated Glycoprotein-2 Specifically Interacts with
a Range of Bovine and Human Cell Types via
V
3 Integrin*
Mark A.
Gibson
§,
David I.
Leavesley¶, and
Leonie K.
Ashman
From the
Department of Pathology, University of
Adelaide, Adelaide, South Australia 5005 and the ¶ Renal Unit,
Royal Adelaide Hospital and the
Hanson Centre for Cancer
Research, Institute of Medical and Veterinary Science, Adelaide,
South Australia 5000, Australia
 |
ABSTRACT |
Microfibril-associated glycoprotein (MAGP)-1 and
MAGP-2 are small structurally related glycoproteins that are
specifically associated with fibrillin-containing microfibrils. MAGP-2,
unlike MAGP-1, contains an RGD motif with potential for integrin
binding. To determine if the RGD sequence is active, a series of cell
binding assays was performed. MAGP-2 was shown to promote the
attachment and spreading of bovine nuchal ligament fibroblasts when
coated onto plastic wells in molar quantities similar to those of
fibronectin. In contrast, ~10-fold more MAGP-1 was required to
support comparable levels of cell adhesion. The fibroblast binding to
MAGP-2 was completely inhibited if the peptide GRGDSP or
the MAGP-2-specific peptide GVSGQRGDDVTTVTSET was added to
the reaction medium at a 10 µM final concentration.
The control peptide GRGESP had no effect on the interaction. These
findings indicate that the cell interaction with MAGP-2 is an
RGD-mediated event. A monoclonal antibody to human
V
3 integrin (LM609) almost completely
blocked cell attachment to MAGP-2 when added to the medium at 0.5 µg/ml, whereas two monoclonal antibodies specific for the human
1 integrin subunit, 4B4 (blocking) and QE2.E5
(activating), had no effect even at 10 µg/ml. Fetal bovine aortic
smooth muscle cells, ear cartilage chondrocytes, and arterial
endothelial cells and human skin fibroblasts and osteoblasts were also
observed to adhere strongly to MAGP-2. In addition, each cell type was
able to spread on MAGP-2 substrate, with the exception of the
endothelial cells, which remained spherical after 2 h of
incubation. The binding of each cell type was blocked when the
anti-
V
3 integrin antibody was included in
the assay, indicating that
V
3 integrin is
the major receptor for MAGP-2 on several cell types. Thus, MAGP-2 may
mediate interactions between fibrillin-containing microfibrils and cell
surfaces during the development of a variety of tissues.
 |
INTRODUCTION |
MAGPs1 are a two-member
family of small structurally related glycoproteins, MAGP-1 (31 kDa) and
MAGP-2 (25 kDa), which are specifically associated with
fibrillin-containing microfibrils (1, 3). Fibrillin-containing
microfibrils (10-12-nm diameter) are important structural components
of the extracellular matrix of most connective tissues. In elastic
tissues such as arteries, lung, and elastic ligaments, these
microfibrils are components of elastic fibers, in association with the
elastic protein, elastin. Fibrillin-containing microfibrils can also
occur as elastin-free bundles in tissues such as ocular zonule,
skeletal muscle, and kidney glomerulus (2-4). The major structural
components of these microfibrils are rod-like 350-kDa glycoproteins
named fibrillin-1 and -2, which appear to be arranged as parallel
bundles of 4-8 molecules joined in series in a head-to-tail manner
(5-8). Fibrillin-1 and -2 have distinct, but overlapping,
spatiotemporal tissue distributions, indicating that the microfibrils
have structural and functional heterogeneity (9-10). It is unclear if
fibrillin-1 and -2 form separate populations of microfibrils or if they
can coexist in the same microfibril. Mutations in the genes for
fibrillin-1 and -2 have been linked to the heritable connective tissue
disorders Marfan syndrome and congenital contractural arachnodactyly,
respectively (11, 12).
Despite being the subjects of intensive investigation, the precise
molecular composition, architecture, and structural and functional
heterogeneity of the microfibrils are still being elucidated (3, 5). An
increasing number of proteins have been identified in association with
fibrillin-containing microfibrils. In addition to MAGP-1 and MAGP-2,
these include microfibril-associated proteins 1, 3, and 4 (13-15);
latent transforming growth factor-
1-binding proteins (16-18);
fibulins (19); and emilin (20). In most instances, it is unclear if the
protein forms part of the microfibril or is adhered to its surface.
There is strong biochemical and immunoelectron microscopic evidence
that MAGP-1 and MAGP-2 are covalently linked by disulfide bonding to
fibrillin-containing microfibrils within tissues (21). MAGP-1
co-distributes with most, if not all, fibrillin-1-containing microfibrils and is localized in a specific periodic manner on the
beads of the "beads-on-a-string" structure of these microfibrils revealed by the rotary shadowing technique (4, 22, 23). This suggests
that MAGP-1 may be an integral component of microfibrils of this type.
MAGP-2 exhibits more restricted tissue and developmental patterns of
distribution, suggesting that MAGP-2 has a more specialized role in
microfibril biology (22). Cloning of MAGP-1 and MAGP-2 revealed that
they each contain a characteristic central motif with close sequence
similarity between the molecules, including precise alignment of seven
cysteine residues (1, 24). It is considered likely that this
cysteine-rich region is involved in the interactions of MAGPs with
other components of the microfibril. In contrast, the other regions of
MAGP-1 and MAGP-2 were found to be very divergent in structure.
Evidence indicates that the N-terminal region of MAGP-1 contains
binding sequences for tropoelastin and type VI collagen, and it is
possible that the glycoprotein functions on the surface of the
microfibrils, as an elastin-binding protein during elastinogenesis and
as an anchoring protein mediating the interaction of
fibrillin-containing microfibrils and type VI collagen microfibrils
(25-27). In contrast, MAGP-2 lacks these binding characteristics,
consistent with MAGP-2 having a function different from that MAGP-1.
The N-terminal region of MAGP-2 was found to contain an RGD motif (1),
suggesting that the glycoprotein may have integrin binding activity
(28, 29). Using an in vitro cell binding assay, we have now
shown that MAGP-2 interacts with a wide range of cell types in an
RGD-dependent manner via
V
3 integrin.
 |
EXPERIMENTAL PROCEDURES |
Materials--
MAGP-1 and MAGP-2 were prepared from fetal bovine
nuchal ligament as described previously (21). The RGD-containing
synthetic peptide MP25A (GVSGQRGDDVTTVTSET, corresponding to amino
acids 25-41 of the deduced primary structure for bovine MAGP-2) was prepared by Chiron Mimotopes (Melbourne, Australia). Other peptides and
bovine fibronectin were purchased from Life Technologies, Inc.
Anti-human
V
3 integrin monoclonal
antibody LM609 (30) was purchased from Chemicon International, Inc.
(Temecula, CA). Anti-human
1 integrin monoclonal
antibodies 4B4 (31) and QE2.E5 (32) were obtained from Beckman Coulter
Inc. and Dr. R. J. Faull (University of Adelaide), respectively.
Anti-
1 integrin antibody P5D2 (33) was a kind gift from
Dr. E. A. Wayner (Fred Hutchinson Cancer Research Center, Seattle, WA).
Nuchal ligament fibroblasts, ear cartilage chondrocytes, and aortic
smooth muscle cells were grown from tissues of 210-day-old bovine
fetuses using the explant technique. Endothelial cells were obtained
from the umbilical arteries of 210-day-old bovine fetuses using the
method of Wall et al. (34). Human skin fibroblasts obtained
from normal adult subjects have been described previously (35). Human
osteoblastic cells, grown from trabecular bone explants of normal
adults, were a kind gift from Dr. D. Haynes (Department of Pathology,
University of Adelaide). All of the above cell types were passaged less
than four times before use in the binding experiments. Human
mononuclear cells were freshly prepared from peripheral blood using a
standard Ficoll density gradient method. Human cell lines that were
used in the binding assay included MO7e (megakaryoblastic) (36), NALM-6
(null lymphoblastoid) (37), BALM-1 (B lymphocytic) (38), and T47D
(breast carcinoma) (American Type Culture Collection).
Cell Adhesion Assay--
The cell adhesion assay was based on that
of Sakamoto et al. (39). Cell culture-grade microtiter
plates were coated with MAGP-1, MAGP-2, or fibronectin (0-200 ng/well)
for 18 h at 4 °C, followed by blocking with BSA (1 mg/ml) in
phosphate-buffered saline for 2 h and rinsing three times with
phosphate-buffered saline for 5 min. For cells that adhere to plastic,
confluent cells were released by treatment with trypsin (0.1%) in
Dulbecco's phosphate-buffered saline containing 0.5 mM
EDTA for 5 min at 37 °C. The suspended cells were washed once with
Dulbecco's modified Eagle's medium containing 10% fetal calf serum
and twice with binding buffer (Hepes-buffered Dulbecco's modified
Eagle's medium containing 1 mg/ml BSA) before being added to the wells
at a density of 2.5 × 104 cells/well in 100 µl of
binding buffer. Cells that form suspensions in culture were suspended
directly in binding buffer. The adhesion assay was conducted at
37 °C for 2 h, and in some instances, the cells were
photographed under phase-contrast using a Nikon Diaphot inverted
microscope. Non-adhering cells were removed by gentle washing with
phosphate-buffered saline. Cell binding activity was measured by assay
for hexosaminidase with p-nitrophenyl
N-acetyl-
-D-glucosaminide substrate at
A405 nm (40).
In some experiments, a synthetic peptide (0.01-50 µM)
was included in the binding reaction. For experiments involving
monoclonal antibodies, the cells were preincubated for 30 min at room
temperature with the antibody (0.02-10 µg/ml), followed by
incubation in the wells at 37 °C for 2 h.
 |
RESULTS |
MAGP-2 Promotes the Adherence and Spreading of a Range of Cell
Types from Fetal Bovine Tissues--
Using a solid-phase cell
attachment assay, MAGP-2 was shown to promote the adherence of nuchal
ligament fibroblasts when microtiter wells were coated with subpicomole
quantities of the protein (Fig. 1A). These amounts were
similar on a molar basis to those required for fibronectin, which
contains an active RGD integrin-binding motif, to promote binding of
the cells. In contrast, 10-fold greater molar quantities of
structurally related MAGP-1, which lacks an RGD motif, were required to
promote equivalent cellular adherence. The adhesion and spreading of
cells was monitored by light microscopy before the removal of unbound
cells prior to the assay (Fig. 1B). Cells in control wells
coated with BSA were observed to be non-adherent and spherical in shape
(Fig. 1B, panel a). In contrast, 0.25 pmol (5 ng)
of MAGP-2 coated onto the wells caused adherence and extensive spreading of the cells (Fig. 1B, panel
b). Cells in wells coated with 0.5 pmol (10 ng) of MAGP-1
showed little evidence of attachment and spreading (Fig. 1B,
panel c). Thus, cell morphology was consistent with the findings of the cell binding assay. Other cell types from
fetal bovine tissues were also tested in the cell binding assay with
MAGP-2. Aortic smooth muscle cells, ear cartilage chondrocytes, and
arterial endothelial cells all showed adhesion to MAGP-2 (Fig. 2). Interestingly, higher quantities of
MAGP-2 were required to support a level of endothelial cell binding
equivalent to that of other cell types, suggesting that endothelial
cell attachment is of lower affinity. This is supported by the
morphological observation that, after the 2-h incubation, the
endothelial cells were adhering, but not spreading, on MAGP-2
substrate. In contrast, the other cell types showed extensive spreading
within this time period (data not shown). The adherence of each cell
type to MAGP-2 was completely blocked by 5 mM EDTA,
indicating that the interaction was a calcium-dependent
process (data not shown).

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Fig. 1.
Nuchal ligament fibroblasts adhere and spread
on MAGP-2. A, MAGP-2 ( ), MAGP-1 ( ), or
fibronectin ( ) was coated in serial amounts on rows of wells of a
microtiter plate. After blocking with BSA, fibroblasts from fetal
bovine nuchal ligament were added and incubated at 37 °C for 2 h. After gentle washing, cell adherence was measured by color
development at 405 nm using a hexosaminidase substrate,
p-nitrophenyl
N-acetyl- -D-glucosaminide. Results are the
means ± S.D. of quadruplicate determinations. B, shown
is cell morphology prior to removal of non-adhering cells. Wells were
coated with BSA only (panel a), MAGP-2 (0.25 pmol/well)
(panel b), or MAGP-1 (0.5 pmol/well) (panel c).
Note the extensive spreading of cells on MAGP-2 substrate.
Bar = 50 µm.
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Fig. 2.
A range of bovine cell types adhere to
MAGP-2. Several cell types from 210-day-old bovine fetuses
(2.5 × 104 cells/well) were incubated in wells coated
with MAGP-2 substrate (0.01-200 ng/well) as described under
"Experimental Procedures." , ear cartilage chondrocytes; ,
arterial endothelial cells; , aortic smooth muscle cells; ,
nuchal ligament fibroblasts. Cell adhesion is expressed as a percentage
of maximal cell binding to the substrate. Results are the means ± S.D. of quadruplicate determinations.
|
|
MAGP-2 Binds to Cells via Its RGD Sequence--
To determine if
the cell adhesion to MAGP-2 was mediated through its RGD sequence, the
binding assay was conducted in the presence of (i) a short peptide
(GRGDSP) that is known to inhibit RGD-dependent binding to
a range of integrins (29), (ii) a 17-amino acid peptide corresponding
to the region of MAGP-2 containing the RGD motif, or (iii) a control
peptide (GRGESP) that has no integrin binding properties (Fig.
3A). Both RGD-containing
peptides were shown to inhibit significantly the binding of nuchal
ligament fibroblasts to MAGP-2 at a peptide concentration of 2.5 µM and to inhibit the interaction completely at a
concentration of 50 µM. In contrast, the control peptide
showed no inhibition of the interaction. These findings were confirmed
morphologically as shown in Fig. 3B. In the absence of
peptide (Fig. 3B, panel a) or in the
presence of the control peptide (panel b), the
fibroblasts adhered and spread extensively on MAGP-2 substrate.
However, in the presence of either RGD-containing peptide (10 µM), the cells failed to adhere and retained a spherical
appearance (Fig. 3B, panels c and
d). Since these RGD-containing peptides specifically inhibited cellular adherence to MAGP-2, it is evident that its RGD
motif was mediating the interaction, most likely via a cell-surface receptor(s) of the integrin family.

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Fig. 3.
RGD-containing peptides specifically inhibit
fibroblast adherence to MAGP-2. A, bovine fibroblasts
from fetal nuchal ligament (2.5 × 104/well) were
incubated in wells coated with MAGP-2 (10 ng/well) as substrate in the
presence of a synthetic peptide at concentrations from 0.005 to 50 µM. , MAGP-2-specific peptide MP25A
(GVSGQRGDDVTTVTSET); , GRGDSP; , the control peptide (GRGESP).
Cell adhesion is expressed as a percentage of the
A405 nm value obtained in the absence of
peptide. Results are the means ± S.D. of quadruplicate
determinations. B, shown is cell morphology prior to removal
of non-adhering cells. Incubations were conducted with no added peptide
(panel a), the control peptide (GRGESP; 10 µM)
(panel b), MAGP-2-specific peptide MP25A (10 µM) (panel c), and GRGDSP (10 µM) (panel d). Note the lack of adherence and
spreading in the presence of RGD-containing peptides.
Bar = 50 µm.
|
|
Specific Inhibition of Cell Adhesion to MAGP-2 by
Anti-
V
3 Integrin Antibody LM609--
A
panel of antibodies known to modulate the binding activity of human
1 and
V
3 integrins was
tested in the cell binding assay with MAGP-2. Preliminary
immunofluorescence studies had shown that each antibody strongly
stained bovine nuchal ligament fibroblasts, with the exception of
anti-
1 integrin antibody P5D2, which did not stain the
cells, suggesting that it did not recognize the bovine form of the
integrin (data not shown). Therefore, antibody 4B4 was used as the
blocking anti-
1 integrin antibody in the cell binding
studies. Experiments showed that the binding of nuchal ligament
fibroblasts to MAGP-2 could be almost completely inhibited by
incubation of the cells with blocking
anti-
V
3 integrin antibody LM609 (at
concentrations above 1 µg/ml) during the binding assay (Fig.
4A). In contrast, antibodies
4B4 and QE2.E5 (blocking and activating antibodies, respectively,
recognizing the human
1 integrin subunit) had no effect
in the binding assay even at a concentration of 10 µg/ml. Consistent
with the above findings, microscopic examination showed that the
fibroblasts treated with the anti-
V
3
integrin antibodies (0.5 µg/ml) were non-adherent and had a spherical
morphology after incubation on MAGP-2 substrate (Fig. 4B,
panel c). This appearance was indistinguishable
from that of control cells incubated in wells lacking MAGP-2 (Fig. 4B, panel a). In contrast, neither of
the anti-
1 integrin antibodies 4B4 and QE2.E5 (10 µg/ml) affected the adherence and spreading of the cells on MAGP-2
substrate (Fig. 4B, panels b and
d). In a separate experiment, both of these antibodies were
shown to modulate the binding of nuchal ligament fibroblasts to
fibronectin (Fig. 5). This confirmed that
both antibodies were functionally active against the bovine
1 integrin subunit and that this receptor is expressed
on the surface of the fibroblasts. Overall, the findings indicate that
fetal nuchal ligament fibroblasts interact with MAGP-2 via
V
3 integrin and that MAGP-2 is not
recognized by the
1 family of integrins.
Anti-
V
3 integrin antibody LM609 also
strongly inhibited the binding of MAGP-2 to the other bovine cell types
that had been demonstrated to adhere to this glycoprotein (Table
I).

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Fig. 4.
Anti- V 3
integrin antibody LM609 specifically inhibits fibroblast adherence to
MAGP-2. A, bovine fibroblasts from fetal nuchal
ligament (2.5 × 104/well) were treated with antibody
at concentrations of 0.002-10 µg/ml and incubated in wells coated
with MAGP-2 (10 ng/well) as substrate. ,
anti- V 3 integrin antibody LM609; ,
anti- 1 integrin antibody 4B4 (blocking); ,
anti- 1 integrin antibody QE2.E5 (activating). Cell
adhesion is expressed as a percentage of the
A405 nm value obtained in the absence of
antibody treatment. Results are the means ± S.D. of quadruplicate
determinations. B, shown is cell morphology prior to removal
of non-adhering cells. Incubations were conducted with no MAGP-2 or
antibody (control) (panel a), no antibody (panel
b), anti- V 3 integrin antibody LM609
(0.5 µg/ml) (panel c), and anti- 1 integrin
antibody 4B4 (10 µg/ml) (panel d). Note the lack of
adherence and spreading in the presence of the antibody to
V 3 integrin. Bar = 50 µm.
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Fig. 5.
Anti-human
1 integrin antibodies 4B4 and QE2.E5
modulate the adhesion of bovine cells to fibronectin. Bovine
fibroblasts from fetal nuchal ligament (2.5 × 104/well) were treated with antibody at concentrations of
0.002-10 µg/ml and incubated in wells coated with fibronectin (100 ng/well) as substrate. , anti- 1 integrin antibody
QE2.E5 (activating); , anti- 1 integrin antibody 4B4
(blocking). Cell adhesion is expressed as a percentage of the
A405 nm value obtained in the absence of
antibody treatment. Results are the means ± S.D. of quadruplicate
determinations.
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To determine the species specificity of cell adhesion to MAGP-2, the
cell binding assay was repeated using a variety of human cell types.
Cell binding assays showed that skin fibroblasts and osteoblasts from
adult human sources also adhered and spread on nanogram amounts of
bovine MAGP-2. The attachment of both cell types to MAGP-2 substrate
was almost completely inhibited by
anti-
V
3 integrin antibody LM609,
confirming that this integrin was also the major mediator of the
interaction with human cells (Fig. 6). A
blocking anti-human
1 integrin antibody (P5D2) had no
effect on the cell binding to MAGP-2. Overall, the results indicate
that MAGP-2 specifically interacts with a range of bovine and human cell types via
V
3 integrin.

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Fig. 6.
Human skin fibroblasts and osteoblasts adhere
to bovine MAGP-2 via
V 3
integrin. Human skin fibroblasts or osteoblasts (2.5 × 104 cells/well) were treated with antibody at
concentrations of 0.001-5 µg/ml and incubated in wells coated with
MAGP-2 (10 ng/well) as substrate. , skin fibroblasts treated with
anti- V 3 integrin antibody LM609; ,
skin fibroblasts treated with anti- 1 integrin antibody
P5D2; , osteoblasts treated with
anti- V 3 integrin antibody LM609; ,
osteoblasts treated with anti- 1 integrin antibody P5D2.
Cell adhesion is expressed as a percentage of the
A405 nm value obtained in the absence of
antibody treatment. Results are the means ± S.D. of quadruplicate
determinations.
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A number of human cell types were found not to adhere to MAGP-2. These
included peripheral blood monocytes and several cancer cell lines,
including MO7e (megakaryoblastic), NALM-6 (null lymphoblastoid), BALM-1
(B lymphocytic), and T47D (breast carcinoma). Interestingly, the BALM-1
cells stained very strongly by immunofluorescence with anti-
V
3 integrin antibody LM609, whereas
the other cell lines stained relatively weakly (data not shown). The
failure of the BALM-1 cells to adhere to MAGP-2 may be attributed to
the integrin being present in an inactive low affinity conformation on
these cells. Modulation of integrin affinity has been extensively
documented (28), and several cell lines of hemopoietic origin have been shown to express integrins on their surfaces in low affinity
conformations (41, 42).
 |
DISCUSSION |
Previous sequence analysis of human and bovine MAGP-2 showed a
conserved RGD-containing sequence (QRGDDVT) to be present in both
proteins (1). Surface probability analysis indicated that the sequence
was likely to reside on the surface of the MAGP-2 molecule. These
observations suggested that the RGD sequence in MAGP-2 might have
integrin binding properties. In this study, we have established that a
variety of human and bovine cell types do specifically adhere and
spread on subpicomole quantities of bovine MAGP-2. Cells that were
found to interact with MAGP-2 include fetal bovine nuchal ligament
fibroblasts, aortic smooth muscle cells, ear cartilage chondrocytes,
and arterial endothelial cells and adult human skin fibroblasts and
osteoblasts. The binding could be almost totally inhibited by
RGD-containing peptides and by a blocking anti-human
V
3 integrin monoclonal antibody. Blocking anti-
1 integrin antibodies showed no effect on cell
binding to MAGP-2. Overall, the results indicate that
V
3 integrin is the major receptor for
MAGP-2 on all of the cell types shown to adhere to the protein.
Integrin
V
3 is the most promiscuous of
the RGD-dependent integrins (29, 43). It has been shown to
mediate the adhesion and spreading of many cell types on a wide range
of matrix macromolecules, including vitronectin, fibronectin,
tenascins, thrombospondin, laminin-1, and osteopontin (44) and, of
particular interest, the microfibrillar proteins fibrillin-1 and -2 (39, 45, 46). Upon ligand binding,
V
3
integrin forms part of focal adhesion complexes, where it can mediate
the interaction of the above matrix components with the
actin-containing cytoskeleton, important for cell spreading and
migration (43). In addition, there is evidence that the integrin is
involved in several other cell signaling pathways (47). Integrin
V
3 has previously been identified on the
surface of each of the cell types found to adhere to MAGP-2 in this
study (39, 48-50). There is some debate about the presence of
significant levels of active
V
3 integrin
on osteoblastic cells. Saito et al. (51) detected only very
low levels of this integrin on cultured human osteoblasts. Gronthos
et al. (49) presented strong evidence that the integrin is
present on cultured osteoblastic cells, but that it might be in a
non-active form. However, more recently, Wendel et al. (52)
showed that the integrin is active on bovine osteoblasts, where it
mediates the interaction of the cells with the novel keratan sulfate
proteoglycan osteoadherin. Therefore, the observation that MAGP-2
interacts with human osteoblasts via
V
3
integrin is consistent with the above study. It is interesting that, in
contrast to other adhering cell types, the arterial endothelial cells
adhered to, but did not spread, on MAGP-2 substrate. This observation
indicates that the cell behavioral consequences of the
V
3 integrin-mediated interaction with
MAGP-2 are not identical for all adhering cell types. This finding is
reminiscent of the study of Joshi et al. (53), who reported
that endothelial cells adhere to tenascin in an
RGD-dependent manner, but do not spread on this substrate.
The authors suggested that the tenascin may be eliciting or enhancing a
signaling function on the endothelial cells. Thus, it is also possible
that the interaction of MAGP-2 and
V
3
integrin on such cells serves predominantly as a signaling mechanism
rather than facilitating the anchoring and spreading function observed
with other cell types.
In previous immunolocalization studies, MAGP-2 has been identified with
fibrillin-containing microfibrils in fetal tissues such as nuchal
ligament, aortic intima, skin, skeletal muscle, and kidney mesangium
(22). These MAGP-2-staining microfibrils were often observed closely
adjacent to the surfaces of cells. Therefore, it is likely that MAGP-2
can interact in vivo with several of the cell types examined
in this study. It should be noted, however, that MAGP-2 also bound to
fetal aortic smooth muscle cells and chondrocytes even though the
protein was not identified in the corresponding tissues (22). In
developing nuchal ligament, the period of maximal MAGP-2 expression
corresponds to the period of highest fibrillin-1 expression that, in
turn, correlates with the early stages of elastinogenesis (22, 54). In
this period, nascent elastic fibers consist of bundles of
fibrillin-containing microfibrils coated with newly forming elastin,
lying closely adjacent and parallel to the extracellular surface of
cells (2). Thus, it has been proposed that the cell directs the
deposition and orientation of the elastic fiber components during
elastinogenesis, resulting in elastic fibers with tissue-specific
morphology, e.g. thick fibers in nuchal ligament,
fenestrated lamellae in aorta, and a range of structures from thin
elastic fibers to elastin-free microfibrillar bundles in skin (2,
3).
To achieve tissue-specific morphology, these processes are likely to be
modulated by variation in the expression and function of intracellular,
cell surface-associated, and extracellular proteins. At the cell
surface, the elastin receptor has been implicated in elastic fiber
assembly (55, 56), and it has been suggested that
V
3 integrin may also be involved in the
process through its interaction with fibrillin-1 (39). Integrins have
already been found to play major roles in the cell-surface organization of fibronectin polymers (57). The evidence presented here indicates that MAGP-2 may also be involved in cell-surface interaction with fibrillin-containing microfibrils. The presence of an active RGD integrin-binding site on this relatively small microfibril-associated protein suggests that interaction with
V
3
integrin is a major function of the molecule. It is interesting that
the location of this RGD motif corresponds to the putative elastin/type
VI collagen-binding site on its structural relative MAGP-1, considered to be important for microfibril/matrix interactions (27). The above
points, together with the restricted tissue distribution of MAGP-2,
suggest that the molecule is involved in modulation of microfibril
interactions with cell surfaces in particular tissue environments.
Since MAGP-2 has been identified in adult kidney mesangium (22), it is
evident that the protein has some enduring role in microfibril biology.
Rather than being a constitutive structural component of all
microfibrils, MAGP-2 may be important for the generation and
maintenance of microfibrils and elastic fibers with tissue-specific
morphological, physical, and cell biological properties. An intriguing
possibility is that, during the above processes, the presence or
absence of MAGP-2 on the surface of the microfibrils may influence the
interaction of cell-surface
V
3 integrin
with fibrillin-1 within the microfibrils, leading to distinct
biological consequences.
 |
ACKNOWLEDGEMENT |
We are indebted to Denise Yates for skilled
technical assistance.
 |
FOOTNOTES |
*
This work was supported by the National Health and Medical
Research Council of Australia.The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed. Tel.: 61-8-8303-5337;
Fax: 61-8-8303-4408; E-mail:
mgibson{at}medicine.adelaide.edu.au.
 |
ABBREVIATIONS |
The abbreviations used are:
MAGPs, microfibril-associated glycoproteins;
BSA, bovine serum albumin.
 |
REFERENCES |
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Gibson, M. A.,
Hatzinikolas, G.,
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