(Received for publication, May 15, 1995; and in revised form, August 14, 1995)
From the
Osteopontin (OPN) is an extracellular matrix protein that binds
to integrin . Here we demonstrate
that two other integrins,
and
, are also receptors for OPN. Human
embryonic kidney 293 cells adhere to human recombinant osteopontin
(glutathione S-transferase-osteopontin; GST-OPN) using
integrin
. When the 293 cells are
transfected with the
subunit, they can also adhere to
GST-OPN using integrin
. Divalent
cations regulate the binding of GST-OPN to both
and
. Mg
and
Mn
support the binding of GST-OPN to these integrins
but Ca
does not. The highest affinity is observed in
Mn
. In the presence of this ion, the affinity of
GST-OPN for
is 18 nM and
the affinity for
is 48 nM.
The antibody 8A2, which is an agonist for
, promotes
the adhesion of 293 cells to GST-OPN even when Ca
is
present. This observation suggests that cellular events could modulate
the affinity of
for OPN.
Collectively, these findings prove that integrins
,
,
and
have similar affinity for OPN.
Therefore, all three integrins must be considered when evaluating the
biological affects of OPN.
Osteopontin (OPN) ()is a secreted phosphoprotein that
was originally isolated from bone(1) . OPN is also found in
many other fluids and tissues including milk, urine, placenta, kidney,
leukocytes, smooth muscle cells, and some tumor cells (for reviews, see (1) and (2) ). OPN supports cell adhesion through its
Arg-Gly-Asp (RGD) integrin recognition motif. OPN is also rich in
aspartic acid residues, and can be heavily glycosylated. The acidic
nature of OPN probably accounts for its ability to modulate the growth
of calcium crystals in both bone (1, 2) and
urine(3) .
Integrin is
the established receptor for OPN. In bone,
is expressed on osteoclasts and it initiates bone resorption by
mediating adhesion of the osteoclast to OPN in
bone(4, 5, 6) . It has also been hypothesized
that OPN and integrin
facilitate
vascular remodeling because these two proteins are co-localized in
smooth muscle cells following balloon angioplasty(7) . Both OPN
and integrin
are also present in
human placenta(8, 9) , so their interaction could also
be relevant to pregnancy.
Although is clearly a receptor for OPN, many other integrins also bind the
RGD motif (10, 11) and no data have excluded other
integrins as receptors for OPN. Therefore, we hypothesized that other
integrins with the
subunit may also bind OPN. The
purpose of this study was to provide a quantitative biochemical
analysis of the binding between OPN and integrins
and
. We reason that a measure of these
binding affinities will allow a meaningful comparison with the binding
affinity of OPN to
(12) . If
more than one integrin does bind OPN with similar affinity, then much
information attributing adhesion and signaling events entirely to the
interaction between OPN and
should
be re-evaluated.
Vitronectin was purified from human plasma by affinity chromatography on heparin-Sepharose as described(14) .
Bound protein was calculated from the
specific activity of the labeled ligand and the results are presented
as molecules bound per cell, [GST-OPN].
Scatchard plots were derived by plotting
/[GSTOPN]
against
, where
represents [GST-OPN]
/total number of cells.
The binding affinity (K
) of cell surface integrin
for GST-OPN is derived from the slope of this plot. In cases where
blocking antibodies were present, preincubation with the antibodies at
14 °C for 15 min was carried out prior to adding
I-GST-OPN. In cases where binding was stimulated with
8A2, the antibody was added simultaneously with the labeled ligand.
The ability of purified integrin to bind GST-OPN was also measured using a solid phase binding
assay previously described(19) . Purified
was immobilized on 96-well Titertek
microtiter plates at a coating concentration of 50 ng/well. After
incubation overnight at 4 °C, nonspecific protein binding sites on
the plate were blocked with 30 mg/ml bovine serum albumin and 1 mM of the desired divalent cation(s) in TBS (pH 7.4). Radiolabeled
GST-OPN in either 2 mM Ca
or 0.2 mM Mn
was then added to the plate. In control
wells, nonspecific binding was measured in the presence of a competing
RGD peptide. Nonspecific binding was subtracted from the total binding
to yield specific binding. Each data point is a result of the average
of triplicate wells.
Figure 1:
FACS analysis of integrin expression on
kidney 293 cells. A panel of monoclonal antibodies was used to assess
integrin expression on wild-type and -transfected
human kidney 293 cells. Cells were incubated with mouse IgG or with the
noted primary antibodies and then with secondary fluorescein
isothiocyanate-conjugated goat anti-mouse IgG. Following extensive
washing to remove free antibody the cells were analyzed by flow
cytometry. The expression level of each integrin subunit is indicated
by the mean fluorescence intensity. The integrin expression profile of
wild-type 293 cells was analyzed with mAb LM609 against
(A), P3G2 against
(B), 14H4 against
(C), and mAb 1977 against
(D). Following transfection of these cells with the cDNA
for
the expression of the
heterodimer was detected with mAb
P3G2 (E). Cells transfected with the vector pcDNA3 alone
exhibited a profile identical to wild-type 293 cells (not
shown).
Figure 2:
Wild-type 293 cells and
-transfected 293 cells adhere to OPN. The adhesion of
wild-type (open bars) and
-transfected (dark bars) 293 cells to GST-OPN was challenged with a series
of blocking monoclonal antibodies. Cell adhesion to GST-OPN was
performed in the presence of LM609
(anti-
), P1F6
(anti-
), P4C10
(anti-
), the mixture of P1F6 and P4C10, L230
(anti-
), and RGD peptide as a control inhibitor. The
results are expressed as a percentage of control adhesion in the
presence of mouse IgG (control). The data are the mean of triplicate
wells. Error bars denote the standard deviation. This
experiment was performed four times yielding identical
results.
The
adhesion of -transfected 293 cells was also blocked by
the antibody against the
subunit (L230).
Approximately 70% of the adhesion of the
-transfected
cells could be blocked by P1F6, an antibody that interferes with ligand
binding to
. The remainder of the
adhesion (30%) could be blocked by antibody against the
subunit, indicating that the endogenous
contributes to the adhesion of these
cells to OPN. These experiments show that
can also mediate cell adhesion to OPN.
Figure 3:
A
comparison of the effects of divalent ions on cell adhesion to
osteopontin and vitronectin. The adhesion of kidney 293 cells
expressing either integrin (panels A and C) or integrin
(panels B and D)
to either GST-OPN (panels A and B) or vitronectin (panels C and D) was tested in buffer containing
Ca
(
), Mg
(
), or
Mn
(
). The adhesion of the
-transfected cells was measured in the presence of
antibody P4C10 to eliminate any contribution of endogenous
to cell adhesion. Adhesion assays
were conducted as described under ``Experimental
Procedures.'' Each data point is the average of quadruplicate
measurements. This experiment was performed four times yielding
identical results. Additionally, in separate experiments, identical
results were obtained when uropontin was used as immobilized
ligand.
Figure 4:
A
measurement of the binding affinity between GST-OPN and
and
. Isotherms of
I-GST-OPN binding to wild-type 293 cells (A) and
-transfected 293 cells (C) maintained in
suspension were generated. Cells were harvested from tissue culture
flasks as usual and were resuspended in adhesion buffer containing 0.5
mM Mn
. Mn
was chosen to
measure the highest affinity between GST-OPN and the two integrins.
I-GST-OPN of increasing concentration was added to the
cells and the mixture was allowed to incubate with rocking for 70 min
at 14 °C. Bound ligand was separated from free ligand by
centrifugation through sucrose cushions (see ``Experimental
Procedures''). Each point is the average of triplicate data points
and each isotherm is representative of at least three repetitions. The error bars show the standard deviation. To derive the affinity
of the interaction between GST-OPN and integrin
or integrin
the data shown in panels A and C were replotted according to the method of Scatchard (53) . This derivation yields Scatchard plots for GST-OPN
binding to
(B) and
(D). The R
values for these lines are 0.87 and 0.90,
respectively.
Figure 5:
Integrin
is also a receptor of osteopontin. A, the binding of GST-OPN to integrin
was also determined by a solid phase
binding assay. This study was done in buffer containing Mn
(0.2 mM,
) or Ca
(2 mM,
) as divalent cation. The binding assay was performed as
described previously(19) . The data are the average of
triplicate points in which the error was less than 12% of the total
binding. Nonspecific binding was less than 8% of the total binding as
determined by incubation with competing RGD peptide. Nonspecific
binding is subtracted from the total binding. B, to ensure
that no contaminating
was present in
the
preparation, an enzyme-linked
immunosorbent assay was performed. The monoclonal antibody 6B9 (
) (18) was used as a probe of integrin
and antibody LM609 (
) was used
to detect integrin
.
Figure 6:
Antibody 8A2 stimulates 293 cell adhesion
to OPN in Ca. A, the adhesion of wild-type
293 cells to GST-OPN was measured in the presence of a range of mAb 8A2
(
) or normal mouse IgG (
). Cells were resuspended in
adhesion buffer containing 2 mM Ca
. The
cells (100 µl at 1.5
10
cells/ml) were allowed
to adhere to GST-OPN at a coating concentration of 10 nM. The
data are the mean of triplicate wells. Error bars denote the
standard deviation. This experiment was performed three times yielding
identical results. B, the affinity and number of binding sites
on 293 cells for mAb 8A2 was measured by generating a binding isotherm
with radiolabeled 8A2. Nonspecific binding was determined by
competition with an excess of unlabeled 8A2 and was typically less than
10% of total binding. The specifically bound counts are shown. C, these data were transformed into a Scatchard plot (53) to quantify the binding affinity and the number of binding
sites.
We also examined the ability of mAb 8A2 to
stimulate cell adhesion across the range of coated GST-OPN (Fig. 7A). In the presence of mAb 8A2, the coating
concentration of GST-OPN that support half-maximal cell adhesion is
similar to that obtained in Mn (Fig. 3A), indicating that both 8A2 and
Mn
induce the high affinity state of
. To verify that mAb 8A2 stimulates
adhesion to OPN by enhancing the affinity state of
, adhesion assays were done in the
presence of mAb 8A2 and a series of antagonists, including RGD peptide,
antibody P4C10 (anti-
), and mAb L230
(anti-
). The adhesion to GST-OPN that is induced by
mAb 8A2 can be blocked by each of the above inhibitors (Fig. 7B). Neither random peptide nor mouse IgG
affected cell adhesion. Several other control experiments were also
performed. These studies showed that the Fab fragment of mAb 8A2 was as
effective as the whole antibody and that mAb 8A2 did not induce the
expression of more
on the cell
surface.
Figure 7:
mAb
8A2 simulates adhesion to GST-OPN through integrin
. A, the adhesion of
wild-type 293 cells to GST-OPN was measured in the presence of a range
of coated GST-OPN in the presence of 1 µg/ml of either 8A2 (
)
or normal mouse IgG (
). Cells (100 µl at 1.5
10
cells/ml) were resuspended in adhesion buffer containing 2 mM Ca
and were allowed to adhere to a range of
GST-OPN for 45 min at 37 °C. The data are the mean of triplicate
wells. Error bars denote the standard deviation. B,
to confirm that integrin
is
mediating 8A2-stimulated adhesion to GST-OPN in Ca
,
the adhesion was challenged by synthetic peptides and monoclonal
antibodies. These are: mAb 8A2 only (A), 100 µM GRGDSP (B), 100 µM SPDGRG (C),
1:500 dilution of anti-
ascites P4C10 (D), 20
µg/ml of anti-
mAb L230 (E), and 20
µg/ml nonspecific mouse IgG (F).
Many interactions between cells and the extracellular matrix
depend on cellular recognition of the RGD motif within adhesive
proteins. Small peptides with the RGD sequence will bind to several
integrin adhesion receptors, but larger adhesive proteins display
considerable integrin binding specificity. Therefore, an important
issue with every RGD-containing adhesive protein is to identify its
receptor(s). OPN, for instance, binds to integrin
, but not to the platelet integrin
(12) . However, it is now
apparent that several integrins have ligand binding properties similar
to
, these are the four other
integrins containing the
subunit,
,
,
, and
(22) . Like
, two of these integrins,
and
, bind to vitronectin. This
functional similarity lead us to suspect that both of these integrins
may also bind OPN. Since both
and
have been identified in tissues,
like bone and the vasculature where OPN is involved in tissue
remodeling(1, 2, 31) , there is the potential
for a physiologically relevant interaction between these integrins and
OPN.
Ideally experiments designed to characterize the interactions
between integrins and their ligands would provide a quantitative
measure of these interactions so that a hierarchy of binding affinities
is available. Here, the affinity between OPN and integrin
and
was determined by measuring the binding of
I-GST-OPN to these integrins present on the surface of
kidney 293 cells. Scatchard analysis shows that in the highest affinity
state, the K
of GST-OPN is 18 nM for
and 48 nM for
. We also measured the apparent
affinity between GST-OPN and purified integrin
. It was impossible to determine a K
using Scatchard analysis because GST-OPN binding
to
immobilized in microtiter wells
was non-dissociable. This non-dissociable binding has been observed
previously with integrin
and its
potential physiologic significance has been discussed(23) .
Despite this binding anomaly, the apparent K
(20
nM) between GST-OPN and purified integrin
is comparable to the affinity
between GST-OPN and purified
measured in the same assay under the same
conditions(12) . In addition, several cell adhesion experiments
showed that the coating concentration of GST-OPN necessary for
half-maximal cell adhesion through
,
(Fig. 3, A and B), and
(12) was
identical. Collectively, our data suggest there is no substantial
preference in the binding of OPN to any of these
-integrins. It is important to reiterate that OPN does
not bind to all integrins. We recently measured the binding of OPN to
the platelet integrin
and showed
that these two proteins do not interact(12) .
The binding of
OPN to its different -integrin receptors is also
similar with respect to divalent ion requirement. We previously found
that both Mg
and Mn
support OPN
binding to integrin
, but that
Ca
suppresses this interaction(12) . Here, we
show that Ca
also fails to support the binding of OPN
to integrins
and
. This observation is important
because it illustrates a key difference between the binding of OPN and
vitronectin to
-integrins. Although small differences
exist in the rank-order potency of divalent ions in supporting adhesion
to vitronectin, physiologic levels of Ca
supported
maximal cell adhesion to this protein through
and
. This is in contrast to the adhesion
to OPN which is not supported at any level by Ca
. In
this regard it is worth noting an important biochemical distinction
between vitronectin and OPN. The vitronectin used in these studies is a
multimer, often containing between 12 and 15 vitronectin moieties per
multimer(32, 33) . There is substantial evidence that
the multimeric vitronectin is also present in extracellular matrices in vivo(32, 33, 34) . In contrast,
the OPN used in these studies was proven to be monomeric by mass
spectral analysis (12) and gel filtration chromatography (data
not shown). The soluble OPN found in body fluids is also assumed to be
a monomer. Consequently, it is possible that multimeric vitronectin
engages several integrins simultaneously, thereby overriding an
otherwise lower affinity between vitronectin and
-integrins in calcium ion.
While Ca does not support OPN binding to integrins
and
, Mn
is able to
enhance the binding. This result is not unexpected because
Mn
is known to activate ligand binding functions of
many
integrins(22, 35, 36, 37, 38) .
The physiologic activation of integrins can also be mimicked by
monoclonal
antibodies(16, 39, 40, 41) . For
example, several studies have demonstrated that integrins can be
subject to physiologic activation. The best example is the platelet
fibrinogen receptor integrin
,
which exists in a dormant state on resting platelets. This integrin
responds to platelet activation by increasing its affinity for soluble
fibrinogen (42) . This increased binding affinity enables
platelet aggregation at the site of a wound. Our data indicate that the
binding of GST-OPN to integrin
can
be enhanced by both Mn
and the mAb 8A2, which is
known to be an agonist of other
-integrins. Although
several other integrins are known to have agonists other than divalent
ions(16) , to our knowledge, this is the first demonstration
that the affinity of an
-integrin can be modulated by
an agonist besides Mn
. By analogy with other
integrins that are similarly stimulated, it is possible that this
artificial stimulus indicates the potential for enhancing the affinity
state of the integrin by physiologic means. It is important to
emphasize that even when Ca
is present, the mAb 8A2
was able to enhance cell adhesion to OPN to maximal levels. Thus, the
suppressive effects of Ca
can be overridden by other
stimuli. In future studies, it will be important to determine if
and
can be similarly stimulated to bind OPN when Ca
is present and to determine if there are cellular signals that
can promote adhesion to OPN in Ca
.
The binding of
OPN to and
may be important to bone
homeostasis. OPN is thought to be one of the most important matrix
proteins for osteoclast adhesion(2, 4) . In addition,
soluble OPN stimulates intracellular signaling in osteoclasts,
including Ca
fluxes and the phosphorylation of
intracellular proteins(43) . It has been reported that integrin
is present on human osteoclasts (44, 45, 46, 47) and that integrin
is present on chicken osteoclast
precursors(48, 49) . Therefore both of these integrins
are positioned to mediate interactions between OPN and cells in bone.
Our finding that integrins
and
have high affinity for OPN indicates
that interactions between OPN and these receptors may play an essential
role in bone remodeling. Blocking the activity of
with antibodies inhibits bone
resorption, but no analogous study has been done with antagonists of
other
-integrins. Our data suggest that similar
experiments should be done with antagonists of
and
.
Recent study also indicates that OPN is involved
in vascular injury and repair(6, 31) . One of the
initial responses to vascular injury is the formation of a neointima
which precedes the formation of atherosclerotic lesions(50) .
Giachelli et al.(51) recently showed that OPN
expression is increased substantially in both rat and human smooth
muscle cells surrounding a vessel that has been exposed to a
catheter-induced injury. Because of the temporal regulation of OPN
synthesis following this insult, the hypothesis was put forth that the
OPN expressed by smooth muscle cells may be an important modulator of
cell migration and proliferation associated with neointima formation (7, 52) . The same group showed that, integrin
mediates only a portion of smooth
muscle cell or to OPN; a major component of this adhesion was not
blocked by antagonists specific for
(7) . The data presented in
this report indicate that integrins
and
should be considered as
candidate OPN receptors involved in guiding vascular repair.
The
kinetic data in this report provide information essential to an
understanding of the biology of OPN. Many adhesive and signaling events
are tied to cellular exposure to OPN. In large part, it had been
assumed that these events are mediated by integrin
because it was the only known OPN
receptor. In conjunction with our prior study(12) , the data in
this report show that
,
, and
have similar affinities for OPN and that the ion regulation of
OPN binding to each integrin is nearly identical. Therefore, along with
,
and
must now be considered
receptors for OPN.