© 1995 American Heart Association, Inc.
Articles |
Osteopontin and ß3 Integrin Are Coordinately Expressed in Regenerating Endothelium In Vivo and Stimulate Arg-Gly-Asp–Dependent Endothelial Migration In Vitro
From the Department of Pathology (V.L., S.M.S., C.M.G.), University of Washington, Seattle; the London (Canada) Regional Cancer Centre (A.F.C.); and the Department of Cell Biology (L.L.), Vanderbilt University, Nashville, Tenn.
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Abstract |
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Abstract Osteopontin is an Arg-Gly-Asp (RGD)–containing acidic glycoprotein postulated to mediate cellular adhesion and migration in a growing number of normal and pathological conditions through interaction with integrin molecules. In this report, we have investigated the potential contributions of osteopontin and one of its receptors, the
vß3 integrin, to
endothelial regenerative processes by using both in
vivo and in vitro models. In vivo, uninjured rat arterial
endothelium had undetectable levels of osteopontin and
ß3-integrin mRNA by in situ hybridization. After balloon
catheter denudation, osteopontin mRNA levels correlated temporally and
spatially with active endothelial proliferation and
migration, with the highest levels observed at the wound edge between 8
hours and 2 weeks after injury, declining to uninjured levels at 6
weeks, when regeneration was complete. Osteopontin protein levels, as
determined by immunocytochemistry, paralleled the time course
of mRNA expression. Likewise, ß3-integrin mRNA and
protein levels were substantially elevated in regenerating
endothelial cells but were not detectable in uninjured
or healed endothelium. In vitro, rat smooth muscle
cell–derived and bacterial expressed mouse recombinant
osteopontins both stimulated the adhesion and directed migration of
bovine aortic endothelial cells through interactions
with the vß3 receptor. Structural mutants
of osteopontin confirmed the importance of the RGD domain for both
adhesion and migration of endothelial cells through
vß3. These data suggest important roles
for osteopontin and ß3 integrin in regenerating
endothelium.
Key Words: osteopontin • integrin • endothelium • adhesion • migration
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Many vascular pathologies, including atherosclerosis and restenosis, are thought to be a direct result of injury, leading to loss of vascular endothelial function and/or integrity. Indeed, in animal models of vascular injury, smooth muscle cell proliferation, growth, and tone have all been shown to be modulated by the presence or absence of a functioning endothelial monolayer.1 2 Likewise, molecules elaborated by injured endothelium may be important in the subsequent events leading to cardiovascular disease, perhaps by providing mitogenic, chemotactic, thrombotic, inflammatory, or fibrotic stimuli. Therefore, identifying factors that promote and restore vascular endothelial integrity is of high priority.
Cell-cell and cell-matrix adhesive proteins, including
integrins and their ligands, are considered to play a central role in
maintaining endothelial integrity.3 For
example, maintenance of the normal endothelial
lining involves attachment of cells to one another as well as to the
substratum via adhesive glycoproteins, such as von
Willebrand factor, collagen, laminin, and
fibronectin.4 Large-vessel endothelium
has been reported to express several integrins that can interact with
these molecules, including 2ß1,
3ß1,
5ß1,
6ß1, and
vß3.3 In addition,
vß1,
2ß1, and
5ß1 may participate in
endothelial cell-cell interactions because they
have been localized to regions of cell contacts.5
Considering the increasing evidence that integrins mediate signaling
pathways,6 it is reasonable to predict that interactions
of these receptor/ligand systems are required for the
maintenance of normal endothelial
integrity.
Specific adhesive interactions are also likely to be critical for
appropriate endothelial regeneration after injury. We
recently identified osteopontin, a secreted Arg-Gly-Asp
(RGD)–containing adhesive glycoprotein, as an
endothelial cell product in a subset of vasa
vasorum in human atherosclerotic plaques7 and granulation
tissue.8 This suggested that osteopontin expression might
be a general characteristic of endothelial cells
undergoing morphogenic changes. These findings are particularly
interesting because osteopontin is an adhesive substrate for several
different cell types, including vascular endothelial
cells, and a migratory stimulus for vascular smooth muscle
cells.9 Further investigation of smooth muscle cells has
indicated that osteopontin can interact with at least three different
v-containing integrins,
vß1,
vß5, and
vß3, although its migratory
activity appears to depend on the presence of a functional
vß3 on the cell surface.10 In
the present study, we have used both in vivo and in vitro
experimental systems to investigate the potential contribution of
osteopontin and one of its receptors, the
vß3 integrin, to
endothelial regenerative and morphogenic processes. In
addition, we have begun to identify the specific structural features of
osteopontin required for integrin-mediated adhesion and
migration.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Probes and Antibodies
The rat cDNA clones for osteopontin, 2B7 and ß3 integrin, a gift from Dr Gideon Rodan (Merck, Sharp and Dohme), have been previously described.11 12 The anti-osteopontin antibody, OP199, is a goat polyclonal antibody raised against rat osteopontin, which has been previously described and characterized in detail.9 Purified IgG fractions of OP199 and normal goat plasma were used for immunocytochemistry. F11, a mouse monoclonal antibody directed against rat ß3 integrin (Pharmingen), has been previously characterized.13 SZ21 is a mouse monoclonal antibody directed against the human ß3 integrin (Immunotech Inc), which does not cross-react with rat.14 Mouse monoclonal antibody directed against human
vß3 (LM609)15 was kindly
provided by Dr David Cheresh (Scripps Research Institute) as purified
IgG. Normal mouse and goat IgG at the same concentrations were used as
controls.
In Vivo Arterial Injury in the Rat
Male Sprague-Dawley rats 3 to 4 months old were used
(Bantin and Kingman Inc, Edmonds, Wash). Surgical procedures were
performed under general anesthesia by
intraperitoneal injection of xylazine (2.2 mg/kg)
and ketamine (4.4 mg/kg) (Anased, Lloyd Laboratories). Balloon
catheter denudation of the rat carotid and aorta was performed with
three passes of a 2F Fogarty embolectomy catheter as previously
described.16 In some cases, small areas of denudation were
generated by passing the uninflated balloon catheter into the aorta
(aortic scrapes). Regrowth areas of endothelium were
visualized by intravenous injection of Evans blue (0.3 mL
of 5% solution in saline) 10 minutes before death. Vessels were
perfusion-fixed with 4% paraformaldehyde in 0.1
mol/L phosphate buffer (pH 7.4).
En Face and Häutchen Preparations
Vessels were excised and pinned out on
polytetrafluoroethylene (Teflon) cards.
Samples for in situ hybridization were stored in 4%
paraformaldehyde until use, and samples for
immunocytochemistry were stored in PBS and stained within 24 hours.
After immunocytochemistry or in situ hybridization (see below), the
tissue was stored in 4% paraformaldehyde until
Häutchen preparations of the endothelium were
made as previously described.17
In Situ Hybridization
En face in situ hybridization of vessels was performed as
previously described.18 Antisense or sense osteopontin
riboprobes were made by linearizing the 2B7 plasmid with Bgl
I or Mam I and using T7 (antisense) or T3 (sense)
polymerases, respectively, in the presence of 35S-labeled
UTP (Amersham). For ß3-integrin subunit probes, the
plasmid RIB3 was linearized with EcoRI or
HindIII, and riboprobes were generated with T3 (antisense)
and T7 (sense) polymerases, respectively, as previously
described.12 Riboprobes were hybridized to the tissue at
55°C overnight. Specimens were washed, the Häutchen procedure
was carried out as described, and specimens were dipped in a
1:1 volume of NTB2 (Kodak) emulsion/distilled water. They were
exposed for 2 weeks, developed, and stained with hematoxylin.
Immunocytochemistry
Paraformaldehyde-fixed samples were rinsed
in PBS, and endogenous peroxidase activity was eliminated
by incubation with 0.3% hydrogen peroxide in methanol for 30 minutes.
Nonspecific binding was blocked by incubation with 2% normal rabbit
serum in 1% bovine serum albumin (BSA)/PBS (for OP199 and
normal goat IgG) or 2% horse serum in 1% BSA/PBS (for F11 and SZ21).
All subsequent antibodies were diluted into 1% BSA/PBS. Primary
antibodies were incubated with specimens for 1 hour at room temperature
at the following final concentrations: OP199 and normal goat IgG, 20
µg/mL; F11 and SZ21, 0.7 µg/mL. After they were washed, secondary
antibodies were applied for 1 hour and consisted of biotinylated rabbit
anti-goat IgG or rat adsorbed horse anti-mouse IgG (1:400,
Vector). After the tissue was washed, it was peroxidase-labeled
with an avidin-peroxidase conjugate (ABC Elite, Vector
Laboratories). Antigen was visualized by incubation with the substrate
3,3'-diaminobenzidine (Sigma Chemical Co). Tissues were stored in 4%
paraformaldehyde until Häutchen preparations were
made (see above).
Cell Culture
Endothelial cells were isolated from bovine
aortas as previously described.19 Cultures were maintained
in Waymouth's MB 752/1 medium (GIBCO BRL) containing 0.23% sodium
bicarbonate, 0.35 mg/mL L-glutamine, 1 mmol/L sodium
pyruvate, and 0.01 mmol/L nonessential amino acids supplemented with
10% fetal bovine serum (Hyclone Laboratories) and 100 U/mL each
penicillin and streptomycin (GIBCO BRL). Cultures were fed with medium
containing 10% fetal bovine serum until confluence and passaged by
detaching with 0.05% trypsin. Cells were maintained in a humidified
incubator at 37°C with 5% CO2. For experiments, cultures
showing healthy morphology between passages 4 and 10 were used.
Osteopontin Protein
Osteopontin was purified from the conditioned medium of rat pup
smooth muscle cells as previously described.9 Recombinant
mouse osteopontin and mutant proteins were expressed in
Escherichia coli as glutathione S-transferase fusion
proteins as previously described.20 Site-directed
mutagenesis was performed to produce two mutant proteins in the RGD
region of mouse osteopontin. The RGD region of the whole protein and
the two mutants are diagrammed in Fig 1
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Adhesion Assay
Adhesion assays were performed as previously
described.9 Briefly, substrates were diluted in PBS and
coated onto 96-well plates overnight at 4°C. Coating efficiencies
were determined by quantitative immunoassay of the coated wells by
using the OP199 antibody, which detects all three proteins equally by
Western blotting (C.M. Giachelli, unpublished observation, 1995). Under
these coating conditions, all three mouse recombinant proteins showed
equivalent coating of wells (not shown). Wells were rinsed with PBS,
and nonspecific binding sites were blocked with 100 µg/mL BSA. After
trypsinization and washing, 30 000 cells were added per well. After 60
minutes, wells were rinsed with PBS, and cells fixed with 4%
paraformaldehyde. Cells were stained with toluidine
blue, solubilized in SDS, and quantified by spectrophotometry at 595
nm. Under these conditions, absorbance was found to be proportional to
cell number.9 Data points represent mean±SEM of
quadruplicate wells.
Migration Assay
Migration assays were performed as previously
described9 in a modified Boyden chemotaxis chamber.
Briefly, test substances were diluted to appropriate concentrations in
DMEM containing 200 µg/mL BSA and placed in the bottom wells of a
modified Boyden chemotaxis chamber (Neuro Probe Inc), unless otherwise
indicated. A polycarbonate filter with 8-µm pores was coated with 1
µg/mL fibronectin to allow uniform endothelial
attachment and placed between the test proteins and the upper chamber.
Endothelial cells were plated at 
50 000 cells per
well in DMEM+200 µg/mL BSA and allowed to migrate for 5 hours at
37°C in a humidified chamber. After the incubation period, cells that
had migrated to the bottom of the filter were fixed with methanol and
stained with hematoxylin. Migration was quantified by cell counts of
three random x400 high-power fields in each well, and all groups
were performed in quadruplicate. For testing the effects of a
concentration gradient, osteopontin was also placed in both the top and
bottom chambers or in the top chamber with the cells.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Expression of Osteopontin mRNA and Protein in Normal and Regenerating Endothelium In Vivo
To examine the distribution of osteopontin mRNA in vascular endothelium, in situ hybridization was performed at various time points before and after balloon catheter denudation of the vessel. Fig 2
shows a time course of osteopontin
expression in en face preparations of the vessels. Hybridization of the
antisense probe to normal uninjured endothelium (Fig 2A) was not greater than sense controls (not shown). Four hours after
injury, endothelial cells still did not express
significant amounts of osteopontin mRNA (not shown), although the
smooth muscle cells at this time point were expressing high levels of
osteopontin, consistent with our previous
findings.21 However, at 8 hours after injury, osteopontin
mRNA was strongly induced in endothelial cells (Fig 2B), particularly at the wound edge. This expression in
endothelial cells was maintained at 1 week after injury
(Fig 2C) and 2 weeks (not shown) and was predominantly expressed at the
regenerating wound edge. Six weeks after injury, the levels of
expression were reduced to levels of uninjured
endothelium (Fig 2D).
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Immunocytochemistry was performed to examine osteopontin protein in
normal and regenerating endothelium (Fig 3). In a normal carotid artery, there was only
occasional and slightly positive staining that was detectable by use of
a goat polyclonal antibody against osteopontin, OP1999
(Fig 3A). In contrast, 2 (Fig 3B) or 8 (Fig 3C) days after
endothelial denudation, abundant levels of osteopontin
were found at the regenerating endothelial wound edge.
The positive staining appeared to be closely cell associated and in
some cells appeared enriched in perinuclear regions (Fig 3B),
suggesting localization of newly synthesized protein in the endoplasmic
reticulum and Golgi apparatus. The cells that were
immediately at the regenerating wound edge exhibited the highest
intensity of staining, although positive cells were not limited to this
region. Similar to mRNA levels, osteopontin protein was again at low
levels 6 weeks after balloon catheter injury (Fig 3D).
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Expression of ß3-Integrin mRNA and Protein in Normal
and Regenerating Endothelium In Vivo
One receptor for osteopontin, the vß3
integrin, has been recently implicated as an important migratory
receptor in several cell types, including endothelial
cells.22 To examine the expression of
ß3-containing integrins after endothelial
injury, we performed in situ hybridization and immunohistochemistry by
using ß3 integrin–specific riboprobes or antibodies,
respectively. As shown in Fig 4A,
ß3-integrin subunit transcripts were undetected in
uninjured endothelium. At 4 and 8 hours after injury,
ß3-integrin mRNA was undetected in injured
endothelial cells (not shown). In contrast,
ß3-integrin mRNA transcripts were readily detectable at
16 hours (Fig 4B), 24 hours (not shown), and 8 days (Fig 4C) after
injury in the regenerating endothelium. Elevated levels
of ß3-integrin mRNA were still visible at 2 weeks after
injury, albeit at reduced levels compared with earlier time points (not
shown). By 4 weeks after injury, when endothelial
regeneration was complete, ß3-subunit mRNAs were once
again undetectable (Fig 4D).
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Similarly, ß3-integrin subunit protein was detected in
regenerating endothelial cells (Fig 4E
) in addition to
platelets/platelet remnants after endothelial
injury. The staining of platelets is consistent with the
presence of abundant IIbß3 receptors in
these cells.23 The staining pattern in
endothelium was diffuse and more concentrated at the
regenerating endothelium-denuded surface interface
compared with distal regions surrounding the intercostal artery. In
striking contrast, no staining above background was observed in
uninjured or completely regenerated endothelium (not
shown) or when an isotype-matched anti-human
ß3-integrin antibody (which does not cross-react with
the rat homologue) was used as primary antibody, thus confirming the
specificity of the anti-rat ß3-integrin antibody (Fig 4F).
Purified Osteopontin Stimulates Endothelial
Adhesion and Migration In Vitro
Two sources of osteopontin were tested for their abilities to
stimulate endothelial adhesion and migration. The first
source was native osteopontin derived from pup rat smooth muscle cell
cultures, which we have previously shown to be active in promoting
adhesion of endothelial cells.9 The second
was recombinant mouse osteopontin produced as a glutathione
S-transferase fusion protein in E coli.19 Fig 5A demonstrates that both smooth muscle
cell–derived (native) and recombinant (GSTOPN) osteopontin sources
stimulated endothelial cell adhesion, whereas the GST
control protein had no effect. Similarly, both forms of osteopontin
stimulated cell migration when placed in the lower chamber in a Boyden
chamber chemotaxis assay (Fig 5B). Concentrations of GSTOPN and native
osteopontin yielding half-maximal cell migration were 8 and 10
nmol/L, respectively. In contrast, there was no migration to the
glutathione S-transferase control protein alone or BSA in the lower
chamber (Fig 5B).
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Checkerboard analysis was carried out to determine the
importance of a concentration gradient for the migratory effects of
osteopontin. Smooth muscle cell–derived osteopontin was used in
this analysis (Fig 6A). Maximal cell migration
to osteopontin required a concentration gradient, since placing
osteopontin in both chambers reduced migration by 60%, and minimal
migration occurred when osteopontin was placed in the top chamber with
the cells.
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To determine whether the vß3 integrin
plays a role in endothelial cell migration, we used a
neutralizing anti-vß3 antibody (LM609).
Inclusion of the anti-vß3 antibody at 20
µg/mL inhibited cell migration by >80% (Fig 6B), demonstrating that
endothelial cells use this integrin to interact with
osteopontin. A normal mouse IgG control at the same concentration did
not inhibit cell migration (Fig 6B). Although
vß3 typically interacts with the RGD
sequence in a variety of proteins, recent evidence suggests that there
may be alternate binding sites24 ; therefore, osteopontin
RGD mutants were analyzed for their ability to support
endothelial adhesion and migration.
RGD-Mutant Osteopontins Fail to Support Endothelial
Adhesion or Stimulate Cell Migration
Recombinant osteopontin was made as two mutants in the RGD
tripeptide sequence.20 One mutant had the aspartic acid
residue of the RGD replaced by a glutamic acid residue (GSTRGE), and
the second mutant was a deletion of three amino acids, RGD, which
abolishes the RGD sequence (GSTdelRGD). These mutants were compared
with the whole recombinant protein in adhesive and migratory potential
(Fig 7). Neither mutant protein was able to support
adhesion of endothelial cells, and adhesion in both
cases was similar to the GST control protein (Fig 7A). The differences
in ability of whole protein and mutant osteopontins to support adhesion
were not due to differences in the amount of substrate, since the
proteins had equivalent coating efficiencies by enzyme-linked
immunosorbent assay (not shown). The mutation or deletion of the RGD
sequence also completely eliminated the ability of the protein to act
as a migratory stimulus, and migration to each was not significantly
above the GST control protein alone (Fig 7B).
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Interaction of Osteopontin With vß5
and vß1 Integrins Requires an Intact
RGD Sequence
The studies described above demonstrated that the RGD sequence is
necessary for osteopontin-mediated endothelial
migration via the vß3 receptor. However,
osteopontin has also been shown to interact with two additional
integrins, vß5 and
vß1.10 Both of these have
been reported to be present on endothelial cells in
culture.3 Therefore, we were interested in testing whether
osteopontin interaction with vß5 and
vß1 also requires the RGD sequence. To do
this, we made use of previously characterized human smooth muscle cells
that do not contain the vß3 integrin but
can adhere to osteopontin via the vß5 and
vß1 receptors.10 When these
vß3-deficient cells were tested in an
adhesion assay with the recombinant GST osteopontin proteins (Fig 8), only GSTOPN was able to support adhesion.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Osteopontin is a secreted RGD-containing adhesive glycoprotein that mediates adhesion and migration of various cell types via interaction with
v-containing
integrins. In two previous studies, we observed that
endothelial cells in angiogenic vessels of
atherosclerotic plaques7 as well as granulation
tissue8 stained positively for osteopontin, whereas
nonangiogenic endothelium showed very little
expression. This suggested that osteopontin expression might be a
common response of endothelial cells undergoing
morphogenic change. The present studies show that although
osteopontin levels are low in normal rat endothelium,
osteopontin synthesis is dramatically increased in regenerating
large-vessel endothelium in vivo. In addition, mRNA
and protein levels for the ß3-integrin subunit were
coordinately upregulated. These data suggest that the interaction of
cells with osteopontin and vß3 integrin
may be involved in the reendothelialization
processes critical for arterial repair. The in vitro
studies reported here support this hypothesis and show that osteopontin
is a chemotactic agent for endothelial cells and that
these cells use the integrin vß3 in an
RGD-dependent manner to interact with osteopontin. The abundant production of osteopontin after injury suggests that this protein may function during endothelial regeneration in vivo. What might that function be? The temporal and spatial distribution of osteopontin mRNA and protein is consistent with a contribution to the processes of cell spreading, migration, and/or proliferation. These are the predominant events occurring during repair of large endothelial wounds, as were created in these studies.25 In vitro studies support this idea, as osteopontin has been shown to be an adhesive substrate for a number of cells, including endothelial cells (Reference 99 and the present study). In addition, osteopontin promotes cell spreading26 and stimulates directed migration of bovine aortic endothelial cells in our Boyden chamber chemotaxis assays. It is not known whether osteopontin may be important for endothelial proliferation, but one report has shown a strong correlation of osteopontin expression with the entrance of smooth muscle cells into the cell cycle.27 However, in another study, soluble osteopontin was unable to stimulate smooth muscle cell replication.28 Clearly, in vivo or in vitro neutralizing experiments are required to examine the question precisely.
Selective expression of integrins after injury may help to determine
the nature of cellular responses to osteopontin. We have previously
shown that at least three integrins can interact with osteopontin:
vß3,
vß1, and
vß5.10 Although all three
receptors mediated smooth muscle cell adhesion to osteopontin, only
smooth muscle cells containing vß3 were
able to migrate to osteopontin, implicating this receptor as the major
migratory receptor for osteopontin. Our in vitro studies confirmed that
endothelial cells also used the
vß3 integrin to migrate toward
osteopontin, prompting an investigation of the in vivo expression
patterns of this integrin during endothelial
regeneration. We found that although the ß3-integrin
subunit mRNA and protein were normally low in uninjured
endothelium, levels of both were elevated after injury
in a time course similar to that observed for osteopontin. These data
suggest that the vß3 integrin might be
elevated in a coordinate fashion with osteopontin. However, we were
unable to document the presence of the v subunit or
vß3 complex in our tissues, because
neither cDNA nor antibodies that can specifically detect the rat
homologues are currently available. However, it is likely that the
ß3 protein we detected is heterodimerized to
v, since no known integrin subunit other than
v can dimerize with ß3 in nucleated
cells.23
We were unable to detect significant ß3-subunit
expression in uninjured rat aorta or carotid
endothelium by either in situ hybridization or
immunocytochemistry. This is in contrast to endothelial
cells in human decidua, which have been shown by
immunostaining to contain v and
ß3.29 Immunocytochemical analyses
have also shown the presence of v and ß3
integrin in human pulmonary large-vessel
endothelium,30 but neither subunit was
detectable in human capillary endothelium.
Interestingly, a recent report has shown that although not present
in normal human skin capillary endothelium,
vß3 is a marker of angiogenic
endothelium in dermal granulation tissue and in the
chick chorioallantoic membrane.31 Thus, the difference in
ß3-integrin expression in our studies may reflect a
species difference or perhaps differences in the phenotypic state of
the endothelium in the various vessels examined.
Besides its ability to act as a migratory receptor for
osteopontin10 and vitronectin,22
the vß3 receptor has other activities that
make it an intriguing candidate as a mediator of
endothelial regeneration. It was recently shown that
the expression of vß3 in angiogenic
vessels is related to cell survival of the growing vascular bud, and
inhibition of vß3 with a neutralizing
antibody led to apoptosis of endothelial
cells.32 These data support the idea that
anchorage-dependent survival of many normal cells may be mediated
by signals generated through extracellular proteins interacting with
integrins. The ligand important for these effects, however, has not yet
been identified. In this regard, it is interesting that osteopontin has
also been shown to mark angiogenic endothelium in both
atherosclerotic7 and granulation8 tissue.
Other vß3 ligands include
vitronectin, fibrinogen, von Willebrand factor, and
fibronectin, all of which would be expected to be present in areas
undergoing angiogenesis as well as regeneration. Therefore, another
potential role for osteopontin and vß3 in
regenerating endothelium may be to promote the survival
of endothelial cells and, hence, the regrowth of the
monolayer. Studies using selective antagonists of
osteopontin will be required to address these issues and are currently
in progress.
Although v-containing integrins typically bind to the
RGD tripeptide sequence, recent data suggest that in osteopontin, other
regions of the protein may interact in an adhesive manner with
cell-surface receptors.24 Our results demonstrate that
the RGD sequence is necessary for adhesion and migration of bovine
aortic endothelial cells to osteopontin, similar to
recent data described in both normal fibroblasts and malignant
cells.20 We have extended those observations, however, by
examining the effects of mutant osteopontins on cell adhesion in cells
with defined osteopontin receptors. In this way, we found that
osteopontin adhesion to vß3,
vß5, and
vß1 all depended on a functional RGD
sequence in osteopontin. These studies, however, do not rule out
cell-specific variability or the possibility that other sequences
in osteopontin are also required for functional activity.
It is interesting to note that recombinant osteopontin stimulated greater maximal adhesion and migration of bovine aortic endothelial cells than did native osteopontin derived from cultured smooth muscle cells. The mechanism for this variance is not known, but it is intriguing to speculate that the posttranslational modifications present on the native osteopontin (ie, sialylation and phosphorylation), which are presumably not present on the recombinant protein made in bacteria, might contribute to the observed differences in activity.
We have studied the functions of adhesion and migration to osteopontin, but it is important to note that the structural requirements of other functional properties, such as calcium binding or interaction with extracellular matrix (ECM) proteins, may not be the same. Since osteopontin may act as an "adapter" molecule by simultaneously binding cell-surface and ECM proteins, these structure-function relations are also of interest. Although osteopontin has been reported to bind to collagen,33 fibronectin,34 and osteocalcin,35 it is not known which domains of osteopontin are involved. Binding of osteopontin to calcium, on the other hand, appears to involve polyaspartic acid–rich peptide in the N-terminal half of the molecule36 and not the RGD tripeptide. Clearly, further studies aimed at identifying cell-surface and ECM binding regions in osteopontin that might be important for endothelial cell function are necessary to address these important issues.
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Acknowledgments |
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This study was supported by National Institutes of Health (NIH) grants HL-18645 (Drs Giachelli and Schwartz), HL-47151 (Drs Schwartz and Giachelli), and DK-47659 (Dr Giachelli) and a grant (No. 3129) from the National Cancer Institute of Canada (Dr Chambers). Dr Chambers is a Career Scientist of the Ontario Cancer Treatment and Research Foundation. Dr Liaw was supported by NIH training grant HL-07312.
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Footnotes |
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Reprint requests to Dr Cecilia M. Giachelli, Department of Pathology, SJ-60, School of Medicine, University of Washington, Seattle, WA 98195. E-mail ceci@u.washington.edu.
Received March 23, 1995; accepted June 19, 1995.
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References |
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