Full text of DOI:10.1038/labinvest.3780393

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LABORATORY INVESTIGATION
Vol. 82, No. 1, p. 37, 2002
Copyright © 2002 by The United States and Canadian Academy of Pathology, Inc.
Printed in U.S.A.
Molecular Interactions and Functional Interference
between Vitronectin and Transforming Growth
Factor-␤
Michael Schoppet, Triantafyllos Chavakis, Nadia Al-Fakhri, Sandip M. Kanse, and
Klaus T. Preissner
Department of Biochemistry (MS, TC, SMK, KTP), 3rd Department of Internal Medicine (TC), and Department of
Clinical Chemistry (NA-F), Medical Faculty, Justus-Liebig-University, Giessen, Germany
SUMMARY: Different extracellular matrix proteins have been described as binding proteins for growth factors, influencing their
storage or presentation towards cellular receptors. The multifunctional adhesive glycoprotein vitronectin (VN), which is found in
the circulation and widely distributed throughout different tissues, has been implicated in the regulation of vascular cell functions,
and these activities could be related to interactions with various growth factors. In vitro, soluble VN interfered with transforming
growth factor-␤ (TGF-␤) binding to isolated extracellular matrix and was found to associate with TGF-␤1 and TGF-␤2 as well as
with other growth factors such as vascular endothelial growth factor, epidermal growth factor, or basic fibroblast growth factor
in a saturable manner. In particular, binding of TGF-␤ was maximal for the heparin-binding multimeric isoform of VN, whereas VN
in a ternary complex with thrombin and antithrombin or plasma VN exhibited weaker binding. Plasminogen activator inhibitor-1
(PAI-1) or heparin, but not desulfated glycosaminoglycans, interfered with binding of VN to TGF-␤, and soluble PAI-1 was able
to dissociate VN-bound TGF-␤. Upon limited plasmin proteolysis of VN, only the fragments comprising the intact aminoterminal
portion of VN bound to TGF-␤ as did a synthetic peptide (amino acids 43 to 62), indicating that TGF-␤ and PAI-1 share common
binding site(s) on VN. Although VN did not influence TGF-␤ bioactivity for mink lung epithelial cells, TGF-␤ dose dependently
inhibited both urokinase-receptor as well as ␣v-integrin-dependent adhesion to VN. This activity of TGF-␤ was reminiscent of the
antiadhesive function of PAI-1. In atherosclerotic tissue sections, staining patterns of VN and TGF-␤ indicated their colocalization.
These findings describe VN as a new binding protein for TGF-␤, whereby specific functions of both factors become modulated
by this interaction. (Lab Invest 2002, 82:37–46).
he extracellular matrix (ECM) is a complex struc-
tural network consisting of different collagens,
brun and Baird, 1991; Roghani et al, 1994; Yayon et al,
1991). Other factors, such as vascular endothelial
growth factor (VEGF), which act as paracrine signal
molecules, also associate with heparan sulfate poly-
saccharides by virtue of their heparin-binding proper-
ties (Neufeld et al, 1999). Bound growth factors di-
rectly interact with cells or become trapped within the
ECM until hydrolytic enzymes release the bound
polypeptides that subsequently gain excess to their
target cells. Several proteins bound to the ECM or to
cell surfaces are thought to interact with such growth
factors and serve to deposit and concentrate them
locally. Examples of such interactions are transform-
ing growth factor-␤ (TGF-␤) and thrombospondin
(Murphy-Ullrich et al, 1992), platelet-derived growth
factor-AB or -BB and SPARC (secreted protein, acidic
and rich in cysteine; Raines et al, 1992), or heparin-
binding growth factor-1 and fibronectin (Kan and Shi,
1990).
T
sulfated proteoglycans, and adhesive glycoproteins,
to which various factors are bound, which dynamically
regulate cellular growth, morphogenesis, and differen-
tiation. Among these, growth factors, proteases, and
their specific inhibitors not only influence the molecu-
lar composition of the ECM but organize motility of
cells and their invasiveness. The earliest reports on
tethering of growth factors to the ECM included the
fibroblast growth factor family (Rapraeger et al, 1991),
demonstrating anchoring of these polypeptides to the
polysaccharide component of heparan sulfate proteo-
glycans. In addition, binding of basic fibroblast growth
factor (bFGF) to cell surface-associated heparin-like
molecules is required for binding of the growth factor
to its high-affinity receptor, thus serving as a dual
receptor system for full growth factor activity (Klags-
Another multifunctional adhesive glycoprotein is vi-
tronectin (VN), found in the circulation and deposited
in several tissues, particularly during wound healing in
the vessel wall and the skin (Preissner, 1991). A high
degree of conformational flexibility leads to the gen-
eration of isoforms of the molecule with heparin-
binding (François et al, 1999) and other exposed
domains, which are thought to endow the protein with
Received September 17, 2001.
This study is part of the thesis by Michael Schoppet at the Medical Faculty,
Justus-Liebig-Universität, Giessen, Germany. This study was supported in
part by Grant Pr 327/1-4 from the Deutsche Forschungsgemeinschaft,
Bonn, Germany, and the Wilhelm-Sander-Stiftung, Munich, Germany.
Address reprint requests to: Dr. Klaus T. Preissner, Medical Faculty, Depart-
ment of Biochemistry, Justus-Liebig-Universität, Friedrichstrasse 24, D-35392
Giessen, Germany. E-mail: klaus.t.preissner@biochemie.med.uni-giessen.de
Laboratory Investigation • January 2002 • Volume 82 • Number 1 37

Schoppet et al
multivalent properties, particularly when associated
with the ECM (Bittdorf et al, 1993; Stockmann et al,
1993). Because of its interaction with different recep-
tor systems, such as the urokinase receptor (uPAR) or
integrins, VN serves as a versatile adhesion molecule
at sites of tissue remodeling. For example, leukocyte
adhesion to VN, which is involved in the recruitment of
these cells to the inflamed tissue, is greatly dependent
on uPAR. In this case, urokinase plasminogen activa-
tor (uPA) acts as a proadhesive factor by increasing
the affinity of the uPAR-VN interaction, whereas plas-
minogen activator inhibitor-1 (PAI-1), which is bound
and stabilized by VN (Declerck et al, 1988), serves as
an antiadhesive factor (Kjoller et al, 1997). Indepen-
dent of its antiproteolytic activity, PAI-1 can block
integrin-mediated adhesion to VN as well (Stefansson
and Lawrence, 1996), a process that is important in
the regulation of vascular cell migration. VN and its
fragments as well as integrins with specificity for VN
are found in areas of inflammation and angiogenesis
(Casaroli Marano et al, 1995; Kramer et al, 1993), and
the detection of these components in atherosclerotic
lesions was reported (Mori et al, 1995). Limited prote-
olysis of VN by plasmin converts the ECM protein into
a plasminogen-binding factor (Kost et al, 1992), which
contributes to the dynamics of cell-ECM contacts
(Ciambrone and McKeown-Longo, 1990).
with binding of TGF-␤ to the ECM (Fig. 1). High
concentrations of heparin exhibited a moderate block-
ade of TGF-␤1 binding to ECM.
Binding of VN to Various Immobilized Peptide Growth
Factors and Binding of Different VN Isoforms to TGF-␤
In addition to TGF-␤, several heparin-binding growth
factors including VEGF, epidermal growth factor
(EGF), and bFGF were tested in their ability to bind to
plasma VN in an ELISA system (Fig. 2A). Because
TGF-␤ exists in humans in several isoforms (TGF-␤1-
3), we tested TGF-␤1 and TGF-␤2 for interaction with
VN but found no difference in the binding pattern of
the isoforms. These data revealed that growth factors
varied in their extent of binding to VN, EGF represent-
ing the highest capacity, whereas bFGF was bound
less effectively. Furthermore, different isoforms of VN
(plasma VN, multimeric VN, ternary complex) were
studied for binding to immobilized TGF-␤1. The most
prominent binding was seen with multimeric VN,
whereas the ternary complex and plasma VN exhibited
less efficient binding (Fig. 2B). The binding constants
(derived from double reciprocal plots) were estimated
to be 0.33 nM for multimeric VN, 2.6 nM for the ternary
complex, and 7.3 nM for plasma VN.
Competition of VN-Growth Factor Interactions by
Glycosaminoglycans and by PAI-1
TGF-␤1-3 are cytokines involved in inflammation,
proliferation, angiogenesis, apoptosis, and ECM pro-
duction relevant in vascular regeneration and remod-
eling (Massague, 1998; Munger et al, 1997). Synthe-
To characterize the binding domains of VN involved in
the interaction with growth factors, different glycos-
aminoglycans were studied for their ability to interfere
with binding of VN to immobilized growth factors,
particularly TGF-␤1. Heparin blocked binding of mul-
timeric VN to immobilized TGF-␤1 and EGF (data not
shown) as did SP-54, a highly sulfated and negatively
charged pentasaccharide, whereas dermatan sulfate
and desulfated heparin failed to interfere with binding
sized by cells in
a latent dimeric form and
noncovalently complexed to the latency-associated
peptide (Massague, 1990), latent TGF-␤ must be ac-
tivated to elicit biologic responses or to bind to their
cognate cell surface receptors (Munger et al, 1997).
Latent TGF-␤ is found associated with the latent
TGF-␤-binding protein, which confers binding to the
ECM and seems to associate with uPAR (Godar et al,
1999), leading to possible TGF-␤ activation or uPAR
recycling.
In the present study, we investigated the interaction
of TGF-␤ with VN and could define binding character-
istics in vitro. In addition, we studied possible func-
tional consequences of this physical interaction in
different cell culture systems, and atherosclerotic tis-
sue sections were investigated for colocalization of
both components. The results indicate that binding of
TGF-␤ to ECM-resident VN or VN in the circulation
mutually influences the bioactivity of both factors,
particularly related to cell adhesion phenomena.
Results
Figure 1.
Binding of TGF-␤ to ECM
Binding of transforming growth factor-␤1 (TGF-␤1) to HUVEC-derived extra-
cellular matrix (ECM). After preincubation (from left to right) of plasma
vitronectin (2, 10, and 20 ␮g/ml), fibronectin (2, 10, and 20 ␮g/ml), fibrinogen
(2, 10, and 20 ␮g/ml), heparin (20, 100, and 200 ␮g/ml), or buffer alone (open
bar) with TGF-␤ (0.5 ␮g/ml) for 30 minutes at 37° C, the individual mixtures
were allowed to react with isolated ECM derived from cultured endothelial
cells. Bound TGF-␤ was quantitated by antibody detection, expressed as
absorbance at 405 nm. Data are mean Ϯ SEM (n ϭ 3) of a typical experiment,
out of three similar ones.
Binding of TGF-␤1 to the ECM of cultured endothelial
cells was tested in the absence or presence of various
soluble competitors such as plasma VN, fibronectin,
fibrinogen, or heparin. Although VN inhibited binding
of TGF-␤1 to ECM in a concentration-dependent
manner, fibronectin and fibrinogen did not interfere
38
Laboratory Investigation • January 2002 • Volume 82 • Number 1

Vitronectin–TGF-␤ Interactions
Figure 2.
Binding of soluble plasma vitronectin (VN) to immobilized growth factors and
binding of different vitronectin isoforms to immobilized TGF-␤. A, TGF-␤1 (Œ),
TGF-␤2 (ꢀ), vascular endothelial growth factor (VEGF) (ꢁ), epidermal growth
factor (EGF) (}), basic fibroblast growth factor (bFGF) (□), and, for control,
fibrinogen (F) were immobilized in microtiter wells, and soluble plasma VN at
the indicated concentrations was allowed to bind. B, Different isoforms of VN,
ie, plasma VN (Œ), multimeric VN (}), or the ternary VN-thrombin-
antithrombin complex (ꢁ), were allowed to bind to immobilized TGF-␤1. For
ELISA detection, a monoclonal anti-VN antibody (VN7) and the respective
peroxidase-conjugated secondary antibody were used to quantitate bound VN,
expressed as absorbance at 414 nm.
of VN to growth factors (Fig. 3A). These results indi-
cate that either the heparin-binding site in VN or the
oppositely charged aminoterminus of the protein is
involved in the described interaction. To specify these
observations, PAI-1 was included in inhibition experi-
ments, because the high-affinity binding site of the
inhibitor is located within the acidic aminoterminal
portion of VN. Although the latent form of PAI-1, which
hardly binds to VN, had no effect on the interaction
between multimeric VN and immobilized TGF-␤1 or
EGF (data not shown), active PAI-1 had a similar and
comparable inhibitory effect on TGF-␤ binding as was
demonstrated for heparin (Fig. 3B). No binding of
active PAI-1 to immobilized TGF-␤1 or EGF could be
detected (data not shown), indicating that the PAI-1
binding site in VN was engaged in binding to these
peptide growth factors. Two-chain high molecular
weight kininogen (HKa), which also binds to the ami-
noterminus of VN and competes with PAI-1 for the
same binding site on VN, was tested but did not inhibit
Figure 3.
Vitronectin binding to growth factors: competition by glycosamino-glycans and
plasminogen activator inhibitor-1 (PAI-1). A, Soluble multimeric VN was allowed to
bind to immobilized TGF-␤1, which was preincubated for 15 minutes with buffer
only (}) or 100 ␮g/ml each of heparin (ꢁ), desulfated heparin (□), dermatan
sulfate (Œ), SP-54 (᭛), or (B) buffer only (}), or heparin (100 ␮g/ml) (F), as well
as 5 ␮g/ml each of latent (ꢁ) or active PAI-1 (Œ). Bound VN was quantitated as
described in the legend to Figure 2. C, In the reverse system, binding of soluble
TGF-␤1 to immobilized VN in the absence (}) or the presence of heparin (100
␮g/ml) was performed, whereby heparin was preincubated with immobilized VN
and either left in the binding mixture (Œ) or unbound heparin was washed away
(ꢁ) before addition of TGF-␤. Quantitation of bound TGF-␤ was measured with the
respective antibody detection system and expressed accordingly as indicated for
VN in the legend to Figure 2.
the interaction between TGF-␤ and VN (data not
shown). In a reverse manner, soluble TGF-␤1 was
found to bind to immobilized plasma VN as well, and
Laboratory Investigation • January 2002 • Volume 82 • Number 1 39

Schoppet et al
blocking of growth factor binding could be achieved
by preincubating immobilized VN with heparin before
the addition of TGF-␤. In contrast, no competition was
observed if unbound heparin was thoroughly removed
before TGF-␤ was added (Fig. 3C).
to TGF-␤. Only one peptide of this series, entailing
amino acids 43 to 62, was able to bind soluble TGF-␤1
(Fig. 4C). These results confirm our initial observations
that the aminoterminus of VN constitutes the binding
site for TGF-␤1.
Influence of VN on TGF-␤ Activity
Binding of VN-Derived Fragments and Peptides to
TGF-␤1
To measure the influence of VN on TGF-␤ bioactivity,
different concentrations of active TGF-␤1 were prein-
cubated with multimeric VN and thereafter added to
mink lung epithelial cells (MLEC) transfected with a
TGF-␤-sensitive PAI-1 promotor construct (Fig. 5).
Activation of cells induced by TGF-␤, as measured by
luciferase activity, did not significantly change in the
presence of multimeric VN. We tested different con-
centrations of multimeric VN and different incubation
times, but there was hardly any influence on the PAI-1
promotor-driven induction of luciferase by TGF-␤1,
indicating that VN binding to TGF-␤ did not affect its
interaction to cellular receptor(s) and subsequent sig-
nal transduction events.
To complement these data, binding of TGF-␤1 was
tested to proteolytic fragments of VN derived after
limited digestion with plasmin. Taking into account
that the carboxyterminal portion of the VN molecule
comprising the heparin binding region is most sensi-
tive to plasmin proteolysis, initial fragments of 65-, 61-
to 63-, and 56-kd containing an intact aminoterminus
of VN were generated. Subsequently, the 56-kd frag-
ment was further degraded into fragments of 42 and
35 kd. Only the initial larger fragments, but not these
shorter peptides, were able to interact with TGF-␤ in a
ligand blot assay (Fig. 4, A and B), supporting the
previous results that the aminoterminus of VN com-
prises the binding region for TGF-␤. Synthetic pep-
tides of this region of VN with overlapping sequences
between amino acids 1 to 130 were tested for binding
Dissociation of the VN–TGF-␤ Complex by Soluble PAI-1
Because PAI-1 competes with TGF-␤ for binding to
VN, we investigated whether preformed VN–TGF-␤
complexes can be dissociated by an excess of soluble
PAI-1. TGF-␤ was allowed to bind to immobilized VN,
followed by incubation with either PAI-1 or HKa.
Although HKa did not interfere with VN-growth factor
complexes, consistent with the competition studies
shown before, in a concentration-dependent manner,
PAI-1 was able to release bound TGF-␤ from VN to an
appreciable degree (Fig. 6).
Antiadhesive Activity of TGF-␤ in Cell Adhesion
The adhesion of leukocytic cells to VN is mediated by
uPAR and augmented by uPA to increase the affinity
Figure 4.
Binding of TGF-␤ to VN-derived fragments. Undegraded VN (Lanes 1) and VN
fragments derived from limited plasmin proteolysis for 10 minutes (Lanes 2),
30 minutes (Lanes 3), or
1 hour (Lanes 4) were separated by SDS-
polyacrylamide gel electrophoresis, transferred onto nitrocellulose and (A)
stained by immunoblotting with the anti-S6/7 polyclonal VN antibody, (B)
incubated with soluble TGF-␤1 followed by the addition of a monoclonal
anti-TGF-␤ antibody. In both cases a secondary antibody system and substrate
was used to stain respective bands as indicated in the “Materials and Methods”
section. C, Synthetic peptides (2 ␮g/ml each) of the aminoterminal region (1
to 130) of the VN molecule comprising the indicated overlapping amino acid
(aa) sequences were immobilized using the Covalink system, and TGF-␤1 (100
ng/ml) was allowed to bind. Bound TGF-␤ was quantitated as indicated in the
legend to Figure 3. Values represent mean Ϯ SEM (n ϭ 3) of a typical
experiment, out of three similar ones.
Figure 5.
Influence of VN on TGF-␤ activity. Mink lung epithelial cells expressing the
TGF-␤-sensitive PAI-1 promotor-driven luciferase construct were stimulated
with the indicated concentrations of TGF-␤, preincubated for 30 minutes at
37° C in the absence (black bars) or the presence of multimeric VN (10 ␮g/ml;
grey bars). TGF-␤ induced effects on MLEC were assessed by measuring
photon emission (relative light units) generated from PAI-1 promotor coupled
luciferase expression. The assay was performed in triplicates, mean values Ϯ
SEM of a representative experiment (n ϭ 3) are shown.
40
Laboratory Investigation • January 2002 • Volume 82 • Number 1

Vitronectin–TGF-␤ Interactions
Figure 6.
Binding of TGF-␤ to immobilized VN. TGF-␤ (1 ␮g/ml) was allowed to bind to
immobilized VN (2 ␮g/ml) for 6 hours at 22° C. Plates were then washed and
250 nM PAI-1 (F), 100 nM PAI-1 (ꢁ), or 250 nM two-chain high molecular
weight kininogen (HKa) (Œ) was added at different time points as indicated.
Thereafter, the remaining TGF-␤ was detected by a polyclonal antibody,
followed by a secondary peroxidase-conjugated antibody against rabbit immu-
noglobulins. Specific binding of TGF-␤ is shown as percent of control, which
is represented by the binding of TGF-␤ to VN in the absence of competitors.
Values represent mean values Ϯ SEM of a typical experiment out of three; * p
Ͻ 0.03, ** p Ͻ 0.0005 compared with binding in the absence of PAI-1.
of the uPAR-VN interaction, whereas the adhesion of
MG63 osteosarcoma cells to VN is dependent on
␣v-integrins, as evidenced by inhibition studies with
cyclic RGDfV peptide (Wong et al, 1996). Because
TGF-␤1 bound to the 43 to 62 peptide region of VN,
which is located proximal to the binding sites for PAI-1
and uPAR as well as the integrin recognition motif
RGD, the effect of TGF-␤1 on VN-mediated cell adhe-
sion was tested. BAF-3 (mouse B cells) transfected
with uPAR adhered strongly to VN, and this adhesion
was inhibited by an anti-uPAR monoclonal antibody
R3 (Chavakis et al, 1999). In a similar manner, TGF-␤1
dose dependently inhibited cell adhesion both in the
absence or the presence of uPA (Fig. 7A). Moreover,
␣v-integrin-dependent MG63 cell adhesion to VN was
inhibited by TGF-␤1, an antiadhesive activity that is
comparable to the propensity of PAI-1 to block cell
adhesion (and migration) on VN substrata (Fig. 7B). In
contrast, MG63 cell adhesion to fibronectin, which is
predominantly ␤1-integrin mediated, was not affected
by TGF-␤1, although TGF-␤ may also bind to fibronec-
tin. These latter data indicate that TGF-␤ can act as a
specific antiadhesive factor in different VN-dependent
adhesion systems.
Figure 7.
Effect of TGF-␤1 on VN-dependent cell adhesion. A, The adhesion of urokinase
receptor (uPAR)-transfected BAF-3 cells to VN in the absence (}) or presence
(ꢁ) of 50 nmol/L urokinase plasminogen activator (uPA) without or together
with increasing concentrations of TGF-␤1 as indicated was measured. Data are
expressed as absorbance at 590 nm and are means Ϯ SEM (n ϭ 3) of a typical
experiment out of three; * p Ͻ 0.02, ** p Ͻ 0.002 compared with adhesion
in the absence of TGF-␤. B, The integrin-dependent adhesion of MG63 cells to
vitronectin (dotted bars) or fibronectin (hatched bars) in the absence (Ϫ) or
presence of 2 ␮g/ml TGF-␤, 10 ␮g/ml cyclic RGD peptide, or 10 mmol/L EDTA
is shown. Data are expressed as absorbance at 590 nm and are means Ϯ SEM
(n ϭ 3) of a typical experiment. Similar results were obtained in three separate
experiments;
competitors.
* p Ͻ 0.01 compared with adhesion in the absence of
modes of interaction of TGF-␤ to ECM components
have been described, including thrombospondin
(Murphy-Ullrich et al, 1992), fibronectin (Mooradian et
al, 1989), collagen type IV (Paralkar et al, 1991), or
proteoglycans like decorin, fibromodulin, biglycan,
and betaglycan (Fukushima et al, 1993; Hildebrand et
al, 1994). In addition, ␣2-macroglobulin is thought to
be one of the major carrier proteins for TGF-␤ in
plasma (James 1990), and complex formation of
TGF-␤ isoforms with ␣2-macroglobulin can result in
differential behavior of cells towards the growth factor.
Although MLEC are not altered in their proliferation
when exposed to TGF-␤1 in complex with ␣2-
macroglobulin, complex formation abolishes the effect
of TGF-␤2 in the same assay (Danielpour and Sporn,
1990). In contrast, fibronectin, collagen, and betagly-
can are thought to form non-neutralizing complexes
Discussion
Mechanisms for tethering growth factors to the ECM
are of importance to concentrate them in the immedi-
ate proximity of cells with impact on biologic function
in vivo. Not only binding and sequestration, but also
modulation of growth factor activity, could result from
their interaction with particular ECM components par-
ticipating in the mechanisms of growth factor signaling
(Klagsbrun and Baird, 1991). Besides binding of mem-
bers of the fibroblast growth factor family to heparan
sulfate proteoglycans (Yayon et al, 1991), several
Laboratory Investigation • January 2002 • Volume 82 • Number 1 41

Schoppet et al
with TGF-␤, whereas decorin was described to inac-
tivate growth factor activity (Yamaguchi et al, 1990). In
all cases, however, particular binding regions for
TGF-␤ on the described components were not spec-
ified in detail.
binding motif for TGF-␤. Additional phosphorylation
sites within this region of VN may contribute to the
acidic nature of this TGF-␤ binding site and could
stabilize growth factor binding (Seger et al, 1998). In
fact, VN is a natural substrate for ectokinases, which
phosphorylate the adhesive protein upon platelet se-
cretion and aggregation reactions (Gechtman and
Shaltiel, 1997) with consequences for ligand binding
to VN (Seger et al, 1998). Because of the fact that VN,
PAI-1, and TGF-␤ are stored in platelet ␣-granules
together with highly condensed glycosaminoglycans
and become released upon platelet activation, high
molecular weight complexes between VN and PAI-1
were found (Preissner et al, 1989), whereas no partic-
ular single complex with TGF-␤ alone was discernible
(data not shown). This may be a result of the fact that
PAI-1 (and glycosaminoglycans) are in excess of
TGF-␤ upon platelet secretion reactions and thereby
prevent stable complex formation between VN and
TGF-␤.
For testing the functional consequences related to
the interaction of TGF-␤ with VN in cell culture sys-
tems, a sensitive cellular assay for TGF-␤ bioactivity
was used (Abe et al, 1994). Upon preincubation of
TGF-␤ with multimeric VN there was hardly any sig-
nificant alteration of TGF-␤ activity with respect to
induction of the PAI-1 promotor-driven luciferase con-
struct. Although VN apparently does not influence the
binding of TGF-␤ to its cognate receptor and the
subsequent activation of signal transduction path-
ways, VN may sequester TGF-␤ into certain tissue
compartments and thereby regulate its availability,
activation, or degradation. This contention is particu-
larly relevant in light of recent studies that showed a
cooperation between the binding protein for latent
TGF-␤ (identical to insulin-like growth factor II recep-
tor or mannose-6-phosphate receptor) and uPAR in
TGF-␤ conversion (Godar et al, 1999).
Another consequence of the TGF-␤-VN interaction
is the interference of TGF-␤ in VN-mediated uPAR-
and integrin-dependent cell adhesion, as demon-
strated here. These antiadhesive properties of TGF-␤
may influence inflammation processes as well as
leukocyte recruitment or endothelial cell functions,
because TGF-␤ was shown to interfere with endothe-
lial cell proliferation in a concentration-dependent
manner (Takashima and Klagsbrun, 1996). Concentra-
tions of TGF-␤ required to exert these effects locally
are believed to be available at sites of platelet activa-
tion/aggregation, inflammatory reactions, or neoplas-
tic processes. However, TGF-␤ concentrations in
plasma are still controversially discussed (Grainger et
al, 2000): because large amounts of TGF-␤ are stored
within platelets, which may become released upon
activation at inflamed tissue sites, local TGF-␤ con-
centrations may exceed those in plasma considerably.
As an abundant pool of TGF-␤, in 1 ml of normal
plasma 2ϫ108 platelets have been found to secrete
more than 40 ␮g of TGF-␤ (Jiang et al, 1995), such that
complete platelet activation would give rise to those
local TGF-␤ concentrations used in the present study.
Furthermore, the fact that TGF-␤ serves as a very
Given the fact that a variety of ECM proteins serve
as binding components for growth factors, we tested
binding of TGF-␤ to ECM, which was inhibited by
soluble VN, whereas fibronectin or fibrinogen could
not duplicate this effect. This initial observation al-
ready indicated that VN could serve as a major as yet
unrecognized TGF-␤ binding protein at these sites.
Because neither preincubation of the ECM with a
polyclonal anti-VN antibody nor removal of ECM/VN-
bound PAI-1 altered binding of TGF-␤ to ECM, it
seems likely that TGF-␤ and VN form a complex that
could prevent TGF-␤ from binding to other ligands in
the ECM. In addition to TGF-␤1, other growth factors
such as EGF, VEGF, and bFGF bound VN with variable
efficiency, and this list was recently extended by the
identification of VN as an insulin-like growth factor II
binding protein (Upton et al, 1999). Furthermore, com-
parison of growth factor binding to the different VN
isoforms revealed that multimeric forms of VN (pre-
dominantly present in the ECM and secreted by plate-
lets) exhibit higher affinity than plasma VN for TGF-␤.
Heparin-binding VN multimers are thought to be the
major reactive forms of the adhesive protein in con-
nective tissue or on cell surfaces and may become
deposited in the vessel wall upon vascular injury or
during aging (Dufourcq et al, 1998; Mullins et al, 2000;
Preissner 1991; Van Aken et al, 1997). Thus, binding to
VN can potentially increase the local concentration of
a respective growth factor at the cell-ECM interface
and thereby influences its functions. In the present
context, we concentrated on the interaction between
TGF-␤ and VN for several reasons: (a) both factors are
contained within platelet ␣-granules and become re-
leased upon activation; (b) the VN binding protein
uPAR can associate with the insulin-like growth
factor-II receptor, which is known as the latency
TGF-␤ binding protein (Nykjaer et al, 1998); (c) both
proteins are abundant at sites of tissue remodeling
and may have common target sites on vascular cells.
To further characterize the VN–TGF-␤ interaction,
glycosaminoglycans and PAI-1 were found to com-
pete for binding of VN to immobilized growth factor,
indicative of the participation of the aminoterminal
portion of (multimeric) VN in binding. Since desulfated
heparin and dermatan sulfate failed to compete the
described association, highly ionic interactions be-
tween TGF-␤ and VN, possibly involving the acidic
region of VN (including sulfated Tyr56, Tyr59), may
govern TGF-␤ binding. These conclusions were cor-
roborated by complementary results from limited di-
gestion of VN to produce TGF-␤ binding fragments of
VN as well as from the identification of the peptide
sequence 43 to 62 of VN to directly bind TGF-␤.
Together, these data strongly support our contention
that the portion of the VN molecule downstream of the
RGD epitope, which is in close proximity to (overlap-
ping) binding sites for uPAR and PAI-1, serves as
42
Laboratory Investigation • January 2002 • Volume 82 • Number 1

Vitronectin–TGF-␤ Interactions
potent inducer of PAI-1 production together with the
observation that PAI-1 could mobilize VN-bound
TGF-␤, points to an amplification loop eventually
leading to prothrombotic or fibrotic vascular compli-
cations. Indeed, increased levels of PAI-1 in patients
are associated with a higher risk for developing throm-
botic events (Wiman 1995) or increased resistance
towards thrombolysis, and upon induction of vascular
lesions in PAI-1Ϫ/Ϫ or VNϪ/Ϫ mice, respectively, a
more protected phenotype was observed as com-
pared with wild-type mice (Eitzman et al, 2000; Kon-
stantinides et al, 2001).
Interestingly, codistribution of VN, PAI-1, and TGF-␤
in atherosclerotic tissue sections further provides ev-
idence for the localization of these interacting proteins
at inflamed tissue sites, such that VN may direct the
deposition of the growth factor to tissues under patho-
physiological conditions (data not shown). Together,
these studies point to a new role for VN as a potential
peptide growth factor binding protein in the ECM, the
circulation, or on cell surfaces. The herein defined
interaction of VN with TGF-␤ may help to protect,
sequester, or modulate its activity and can shed light
on unexpected functions of this growth factor with
regard to cellular adhesion.
RGDfV-peptide from Bachem (Heidelberg, Germany),
and fibronectin from Sigma (Munich, Germany).
Antibodies
The monoclonal antibody against TGF-␤1 was ob-
tained from Genzyme (Cambridge, Massachusetts), a
rabbit polyclonal antibody against TGF-␤1-3 was ob-
tained from Santa Cruz Biotechnology (Santa Cruz,
California), and the monoclonal antibody VN7 against
VN was obtained by standard hybridoma techniques
(Stockmann et al, 1993). Monoclonal antibody R3
against uPAR was a kind gift from Dr. G. Hoyer-
Hansen (The Finsen Laboratory, Copenhagen, Den-
mark). The polyclonal antibody anti-S6/7 against VN
was raised in rabbits, and the IgG fraction was isolated
using established procedures (Preissner et al, 1985).
After
modification
of
the
antibody
with
N-hydroxysuccinimido-biotin as described (Preissner
et al, 1989), detection was achieved by peroxidase-
conjugated avidin (Dako, Hamburg, Germany). For
detection of murine monoclonal antibodies, second-
ary goat and rabbit anti-mouse peroxidase-
conjugated antibodies were used (Dako).
Binding of TGF-␤ to ECM
HUVEC were grown to confluency on 96-well culture
plates in DMEM containing 10% fetal calf serum. ECM
was prepared by treating the cell monolayer with 0.5%
(w/v) Triton X-100 and 0.2 M NH3 as described
(Mimuro and Loskutoff, 1989). ECM was subsequently
blocked with 3% BSA in PBS for 1 hour at 22° C,
followed by washing with PBS containing 0.1% (w/v)
Tween 20 (PBS-Tween). Competitors were preincu-
bated with TGF-␤1 for 30 minutes at 37° C before
binding studies, and bound growth factor was de-
tected by the monoclonal anti-TGF antibody and the
respective secondary antibody.
Materials and Methods
Materials
TGF-␤1 and TGF-␤2 were obtained from Berlex
(South San Francisco, California), EGF was kindly
provided by Dr. L. Fryklund (University, Uppsala, Swe-
den), and bFGF was provided by Dr. L. Schweigerer
(University of Essen, Essen, Germany). Fibrinogen was
purified from plasma (Mahn and Muller-Berghaus,
1975), and VN was purified from pooled human
plasma in its native protein conformation as described
(Preissner et al, 1985). To obtain the multimeric form of
VN, plasma VN was incubated with 6 M urea for 1 hour
at 37° C followed by subsequent dialysis against Tris-
buffered saline (TBS; 20 mM Tris, pH 7.4, 150 mM
NaCl) to remove urea (Stockmann et al, 1993). The
ternary VN-thrombin-antithrombin complex was pro-
vided by Dr. H. de Boer (University of Utrecht, Utrecht,
The Netherlands); PAI-1 and plasmin were obtained
from Dr. H. Pannekoek (Academic Medical Center,
Amsterdam, The Netherlands). Active PAI-1 was gen-
erated by addition of 4 M guanidinium chloride for 2
hours at 22° C, followed by dialysis against TBS at
4° C. VN was proteolyzed with plasmin at a VN/
enzyme ratio of 10:1 (w/w) for the indicated time
intervals at 37° C. The reaction was stopped by addi-
tion of SDS-containing sample buffer (Laemmli, 1970).
The VN-derived synthetic peptides comprising over-
lapping sequences of the aminoterminal region of VN
(amino acids 1 to 130) were kindly provided by Dr. A.
Jonczyk (Merck KGaA, Darmstadt, Germany). Single
or two-chain high molecular weight kininogen was
purchased from Enzyme Research Laboratories
(South Bend, Indiana). uPA isolated from natural
sources was from Medac (Hamburg, Germany), cyclic
Binding Assays
Binding of growth factors to VN was studied in an
ELISA system using Maxisorp 96-well plates (Nunc,
Wiesbaden, Germany). Growth factors were coated
for 16 hours at a concentration of 2 ␮g/ml in coating
buffer (15 mM Na2CO3, 35 mM NaHCO3, pH 9.6)
followed by blocking with 3% (w/v) BSA in TBS for 1
hour at 22° C. After three subsequent washes with
PBS-Tween, plasma VN, the ternary VN complex, or
multimeric VN were added in PBS containing 0.05%
(w/v) Tween 20 and 1% (w/v) BSA in a final volume of
50 ␮l for 2 hours at 22° C. After washing, bound VN
was quantified by sequential incubation with the pri-
mary antibody VN7 (1.5 ␮g/ml) or biotinylated anti-VN
(1:5000 dilution) in PBS-Tween for 1 hour at 22° C and
peroxidase-conjugated secondary antibodies. Colori-
metric detection was performed after incubation of
wells with 2,2'-azino-di-(3-ethylbenzothiazoline) sul-
fate and H2O2 in 0.1 M sodium acetate/0.05 M
NaH2PO4 (pH 5.5) in a microtiter plate reader (Bio-
Rad, Munich, Germany) at an absorbance of 414 nm.
For competition experiments, glycosaminoglycans or
Laboratory Investigation • January 2002 • Volume 82 • Number 1 43

Schoppet et al
PAI-1 were preincubated for 15 minutes at twice the
final concentration before the addition of VN. Binding
of soluble TGF-␤1 (2 ␮g/ml) to coated plasma VN was
followed by detection with a monoclonal anti-TGF-␤
antibody (1 ␮g/ml) and a goat anti-mouse peroxidase-
conjugated secondary antibody (1:10,000 dilution).
Before the addition of TGF-␤, immobilized VN was
preincubated with heparin (100 ␮g/ml) for 30 minutes,
with either thorough washing of heparin-containing
wells to remove unbound heparin or no washing.
Binding of VN to noncoated plates showed less than
5% binding compared with growth factor-coated
wells. Data are presented as mean Ϯ SEM of three
independent experiments. For binding studies with
immobilized VN-derived synthetic peptides, the Co-
valink system (Nunc) was used, exactly following the
instructions of the manufacturer. Soluble TGF-␤1 was
added in a final concentration of 500 ng/ml and
detected by the monoclonal anti-TGF-␤ antibody and
the goat anti-mouse peroxidase-conjugated second-
ary antibody.
medium before addition to the cells. After incubation
for 18 hours at 37° C, lysis of MLEC was achieved by
substrate and lysis buffer (Promega, Madison, Wis-
consin) following the instructions of the manufacturer.
Dissociation of VN-Bound TGF-␤ by PAI-1
Binding of TGF-␤ to immobilized VN was followed by
incubation of different concentrations of PAI-1 as
indicated in the legend to Figure 6 to study the release
of VN-bound growth factor. Unreleased TGF-␤ was
quantitated by incubation with a polyclonal anti-TGF-␤
antibody followed by the respective secondary anti-
body as described above.
Cell Culture
BAF-3 (interleukin-3-dependent mouse B-cell line)
cells were obtained from American Type Culture Col-
lection (ATCC; Rockville, Maryland) and cultured in
RPMI-1640 medium containing 10% fetal calf serum
and 2 ng/ml interleukin-3. BAF-3 cells were trans-
fected by electroporation with uPAR complementary
DNA in the sense and antisense orientation using the
expression vector pCDNA3. Cells were selected in the
presence of G418 (1.2 mg/ml) and found to express
uPAR by fluorescence-activated cell sorter analysis,
Northern blotting, and uPAR-enzyme linked immu-
nosorbent assay (Chavakis et al, 1999, 2000). MG63
human osteosarcoma cells were obtained from ATCC
and cultured in DMEM containing 10% fetal calf
serum.
Gel Electrophoretic Analysis and Immunoblotting
SDS-polyacrylamide gel electrophoresis was per-
formed using the buffer system described by Laemmli
(Laemmli, 1970). After electrophoresis at 4° C for 2
hours in a mini-gel apparatus (Bio-Rad), proteins were
transferred to nitrocellulose at 100 V for 2 hours, using
the buffer system of Towbin (Towbin et al, 1979). After
transfer, proteins were blocked by addition of 3%
(w/v) BSA in PBS for 16 hours at 4° C followed by
washing with PBS-Tween. Immunoblotting was per-
formed by incubation with anti-VN (S6/7) as primary
antibody (1:1000 dilution) for 2 hours in PBS-Tween at
22° C followed by incubation with the respective sec-
ondary antibody (peroxidase-conjugated, anti-rabbit
IgG, 1:3000 dilution). For identification of TGF-␤1
binding to plasmin-digested VN fragments by ligand
blotting, TGF-␤1 was incubated in PBS-Tween for 2
hours at 22° C before the addition of antibodies.
Bound TGF-␤1 was detected by a monoclonal anti-
body against TGF-␤1 (1 ␮g/ml) followed by the re-
spective secondary antibody. Bands were visualized
by using the enhanced chemiluminescence method
(Amersham, Buckinghamshire, United Kingdom) ac-
cording to the manufacturer’s instructions. Blots were
exposed to x-ray films (X-Omat AR; Kodak, Rochester,
New York) for 10 to 30 seconds.
Cell Adhesion Assay
Cell adhesion of uPAR-transfected BAF-3 cells to
VN-coated plates was tested according to a previ-
ously described protocol (Chavakis et al, 1999, 2000).
Briefly, multiwell plates were coated with 2 ␮g/ml VN
or 4 ␮g/ml fibronectin, respectively, and blocked with
3% (w/v) BSA. UPAR-transfected BAF-3 cells or
MG63 cells were washed in serum-free RPMI or
DMEM, respectively, and plated onto the precoated
wells for 60 minutes at 37° C in the absence or
presence of competitors in serum-free RPMI or
DMEM, respectively. After the incubation period, the
wells were washed and the number of adherent cells
were quantified by crystal-violet staining at 590 nm.
MLEC-Based Activity Assay for TGF-␤
Statistical Analysis
This assay (Abe et al, 1994) uses a stably transfected
construct of a shortened PAI-1 promotor coupled to a
luciferase reporter gene kindly provided by Dr. D. B.
Rifkin (NYU Medical Center, New York, New York).
Induction of the PAI-1 promotor by soluble TGF-␤
leads to transcription of the luciferase gene and con-
secutively to emission of photons measured by a
luminometer (LB 953 Autolumat; Berthold, Wildbad,
Germany). Multimeric VN was preincubated with ac-
tive TGF-␤1 for 30 minutes at 37° C in cell culture
Triplicate values were compared using the Student’s t
test. Probability values Ͻ0.05 were regarded as
significant.
Acknowledgements
We are grateful to Mr. Thomas Schmidt-Wöll and
Ms. Susanne Tannert-Otto for their skillful technical
assistance.
44
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