From the
Binding of substrate-bound extracellular matrix proteins to cell
surface integrins results in a variety of cellular responses including
adhesion, cytoskeletal reorganization, and gene expression. We have
previously shown that addition of soluble SC5b-9, the
complement-vitronectin complex, resulted in an RGD-dependent increase
in lung venular hydraulic conductivity (Ishikawa, S., Tsukada, H., and
Bhattacharya, J.(1993) J. Clin. Invest. 91, 103-109). To
identify specific integrin(s) and signal transduction pathways that are
responsive to soluble vitronectin-containing ligands, we exposed
confluent bovine pulmonary artery cells to purified soluble human mono-
or multimeric vitronectin, or SC5b-9, and determined the extent of
endothelial cell protein tyrosine phosphorylation. Monomeric
vitronectin (Vn) did not induce enhanced protein tyrosine
phosphorylation. However, multimeric Vn and SC5b-9 elicited time- and
concentration-dependent increases in tyrosine phosphorylation of
numerous proteins. Antiserum against vitronectin, RGD peptides, and
monoclonal and polyclonal antibodies against the
Integrins are cell surface receptor proteins which exist as
transmembrane heterodimers. The extracellular domains of integrins bind
a variety of proteins found in the extracellular matrix (ECM),
Most studies designed to test the capacity of
integrins to promote transmembrane signaling utilize substrate-bound
ligands, thus mimicking their distribution in the ECM. Interestingly,
Conforti et al.(20) demonstrated the presence of the
integrin
Several
integrins capable of binding Vn promote transmembrane signaling in a
variety of cells. For example,
There have been relatively few studies to date reporting
integrin-mediated signaling in endothelial cells. One study
demonstrated
We recently demonstrated that serum
containing the activated complement
The following preparations of Vn were used: native (monomeric) Vn (38, 39) kindly supplied by K. T. Preissner (Haemostasis
Research Unit, Bad Nauheim, Germany) and D. Mosher (University of
Wisconsin, Madison, WI); multimeric Vn, prepared by incubation of
monomeric Vn with 6 M urea followed by dialysis and
lyophilization (supplied by K. T. Preissner); conformationally altered
Vn, purified from human plasma as described by Yatohgo et al.(40) and further purified by gel filtration. To obtain
conformationally altered Vn(40) , we allowed 100 ml of human
plasma to clot in glassware and added 0.2 M phenylmethylsulfonyl fluoride to the resultant serum. After
centrifugation, the supernatant was applied to a Sepharose 4B
precolumn, and then to a heparin-Sepharose column. The flow-through
fractions were subjected to repeated denaturation with 8 M urea, to reduction with
This study demonstrates that soluble Vn-containing ligands
are capable of triggering enhanced protein tyrosine phosphorylation in
bovine pulmonary artery endothelial cell monolayers. Several lines of
evidence suggest that clustering
Vn in its monomeric form is
present in the serum in concentrations similar to those used in this
study. Only a small percentage of Vn isolated from healthy volunteers
is found in high molecular weight complexes, such as ternary complexes
containing thrombin and antithrombin III (reviewed in Ref. 50). The
method of isolation of Vn has considerable influence on its physical
properties. For example, incubation of Vn with 6-8 M urea and
Despite the
exposure of a heparin-binding domain by urea, the heparin-binding
domain did not detectably contribute to the enhancement of protein
tyrosine phosphorylation in BPAECs, whereas the RGD site was required
for this effect. Although these findings seem to conflict with those
showing an inhibition of binding of multimeric Vn to endothelial cells
by heparin(43, 44) , there are several plausible
explanations for this apparent discrepancy. First, the extent to which
heparin inhibits the binding of multimeric Vn to endothelial cells is
dependent on the source of the endothelial cells. Heparin inhibited
more than 90% of the binding of multimeric Vn to human umbilical vein
endothelial cells (43), whereas it only inhibited
It is highly unlikely that luminal
The functions of the particular tyrosine kinase
substrates identified in this study are only partially understood.
Paxillin, a 68-kDa vinculin-binding protein(53) , undergoes
enhanced tyrosine phosphorylation in response to a variety of
agonists(18, 41, 54, 55) . Paxillin has
been shown to bind with high affinity to the SH3 domain of c-Src (56) and v-Crk(57) . Ezrin and cortactin are also
associated with the cytoskeleton, and in fact bind F-actin
directly(58, 59) . Alterations in the cytoskeleton of
endothelial cells have been demonstrated for a variety of stimulii,
including those that mediate alterations in monolayer barrier function
(reviewed in Ref. 60), and preliminary evidence suggests that
alterations in the state of tyrosine phosphorylation affects
endothelial cell barrier function.
It is particularly interesting to note that we demonstrated
enhanced tyrosine phosphorylation of p125
We did not
address in this study the capacity of soluble Vn ligands to mediate
alterations in endothelial cell barrier function. We would predict,
however, that
We thank Rashmi Patel for technical assistance.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
integrin blocked the vitronectin- or
SC5b-9-induced enhanced accumulation of tyrosine phosphoproteins, while
antibodies against
integrins and the
integrin did not. Clustering of the
integrin using monoclonal antibody
LM609 caused a pattern of enhanced tyrosine phosphorylation similar to
that caused by multimeric Vn and SC5b-9, suggesting that aggregation of
was critical for signaling. Among
the proteins that underwent enhanced tyrosine phosphorylation in
response to vitronectin were the cytoskeletal proteins paxillin,
cortactin, and ezrin, as well as the SH2 domain-containing protein Shc,
and p125
. We conclude that ligation of the
integrin by soluble ligands promotes
enhanced phosphorylation of several proteins implicated in tyrosine
kinase signaling and suggest that this pathway may be important in
inflammatory states which are accompanied by accumulation of SC5b-9.
(
)such as laminin, fibronectin, and vitronectin
(reviewed in Ref. 1-4). Upon adhesion to the ECM, cells bearing
integrins demonstrate a diverse array of responses, such as increases
in [Ca
]
(5, 6, 7) and intracellular pH (8),
activation of protein kinase C (9) and mitogen-activated protein
kinase(10) , activation of gene expression (reviewed in Ref.
11), and enhanced protein tyrosine phosphorylation (reviewed in Ref. 2,
12). Among the few substrates identified that undergo enhanced tyrosine
phosphorylation in response to integrin ligation are
p125
(13, 14, 15, 16) ,
pp60
(17) , paxillin(18) , and
tensin(19) .
at the luminal aspect of
endothelial cells in vivo, which suggests that this integrin
may be accessible to soluble stimulii. Although the identity of soluble
ligands for this receptor is unknown, one likely candidate is SC5b-9,
the complement-vitronectin complex which is found in the serum during a
variety of inflammatory states accompanied by activation of complement
(21, 22). Substrate-bound SC5b-9 has been shown to promote the
adherence of myoblasts via Vn contained within SC5b-9 and the
integrin(23) .
mediates elevations in [Ca
]
in osteoclasts (6), enhanced invasiveness and protection
against apoptosis of M21 melanoma cells(24, 25) , and
angiogenesis in response to various agonists(26) . Intravenous
administration of mAb LM609, which blocks binding of ligands to
, promotes regression of several
tumors by inducing apoptosis of angiogenic blood vessels in chick
embryos(27) . In contrast,
mediates endocytosis of Vn in fibroblasts (28) and
migration on Vn-containing matrices in keratinocytes (29) and
pancreatic carcinoma cells(30) . The capacity of
, another Vn-binding
integrin(31) , to initiate transmembrane signaling has not been
reported.
-mediated elevations in
[Ca
]
in human
umbilical vein endothelial cells (5). A more recent study demonstrated
enhanced protein tyrosine phosphorylation in human umbilical vein
endothelial cells migrating on fibronectin, although the precise
receptor(s) mediating this effect was not identified(32) .
Although untested, the ability of the
integrin to mediate enhanced protein tyrosine phosphorylation is
suggested by two lines of evidence. First, binding of fibrinogen to the
platelet integrin
(GPIIb-IIIa),
which is structurally similar to
(33) , leads to enhanced
phosphorylation of several tyrosine kinase substrates(34) .
Second, insulin stimulation of Rat-1 fibroblasts transfected with DNA
encoding the human insulin receptor was shown to induce the association
of the
integrin with several
molecules implicated in tyrosine kinase-mediated signaling pathways,
such as phosphatidylinositol-3 kinase and insulin receptor
substrate-1(35) .
Vn complex SC5b-9 increased
lung endothelial hydraulic conductivity (Lp) through an
integrin-dependent mechanism (36). In this study, we examined the
effects of soluble Vn-containing ligands on the pattern of protein
tyrosine phosphorylation in adherent bovine pulmonary artery
endothelial cell monolayers and identified
as the integrin responsible for mediating enhanced
phosphorylation of several cytoskeletal-associated tyrosine kinase
substrates, as well as p125
.
Cells and Materials
Bovine pulmonary artery
endothelial cells (BPAECs), purchased from American Type Culture
Collection (Rockville, MD), were maintained in Dulbecco's
modified Eagle's medium supplemented with 10% fetal bovine serum,
100 units/ml penicillin, and 100 µg/ml streptomycin. Cells were
passed upon attaining confluence. Their identity was confirmed by
noting typical cobblestone morphology and positive immunofluorescent
staining for Factor VIII antigen. Polyclonal antiserum to SC5b-9, and
an enzyme-linked immunosorbent assay kit to detect SC5b-9, were from
Quidel (San Diego, CA). Polyclonal antiserum against vitronectin and
mAb P1F6 against the integrin were
from Chemicon (Temecula, CA). mAb Z035 against paxillin was from Zymed
Laboratories (San Francisco, CA). Polyclonal and monoclonal antibodies
against Shc were from Transduction Laboratories (Lexington, KY).
Affinity purified rabbit IgG against phosphotyrosyl-containing proteins
was from ICN Biomedicals (Costa Mesa, CA). Horseradish peroxidase- and
alkaline phosphatase-conjugated secondary antibodies were from Jackson
Immunoresearch (West Grove, PA). Sulfosuccinimidobiotin was from
Pierce. Protein A-agarose and Protein A/G PLUS-agarose were from Santa
Cruz (Santa Cruz, CA). Heparin was from Elkins-Sinn (Cherry Hill, NJ),
and peptides were from Peninsula Laboratories (Belmont, CA). Antiserum
838 against the
integrin, and
polyclonal antiserum against
integrins, were
generously provided by S. Albelda (University of Pennsylvania,
Philadelphia, PA). mAb LM609 against the
integrin was kindly provided by D. Cheresh (Scripps Clinic and
Research Foundation, La Jolla, CA). mAb 4F11 against cortactin was
kindly provided by J. T. Parsons (University of Virginia,
Charlottesville, VA). Polyclonal antiserum against p125
and ezrin were generously provided by S. K. Hanks (Vanderbilt
University, Nashville, TN) and A. Bretscher (Cornell University,
Ithaca, NY), respectively.
Purification of SC5b-9 and Vitronectin
SC5b-9 was
purified by the method of Gawryl et al.(37) . In brief,
SC5b-9 was generated by zymosan activation of human serum (37) followed by ammonium sulfate precipitation, polyethylene
glycol fractionation, DEAE-Sephacel chromatography, and lyophilization.
The presence of purified SC5b-9 was confirmed by enzyme-linked
immunosorbent assay. The presence of vitronectin in the complex was
confirmed by SDS-PAGE and immunoblotting using antiserum against Vn.
-mercaptoethanol, and to heparin
affinity chromatography. Samples were dialyzed against PBS and
lyophilized. Verification of purification was assessed by Coomassie
Blue staining of SDS-PAGE under reducing conditions, and further
verified by immunoblotting using polyclonal anti-Vn, which showed the
expected doublet corresponding to a M
of 65 and
75. In some experiments, Vn was fractionated by gel filtration using
either a Bio-Gel P-100 column ((1.6
100 cm); Bio-Rad)
equilibrated with Tris-buffered saline, pH 7.4, containing 0.5%
polyethylene glycol and eluted with Tris-buffered saline, or a
Sephacryl S-300 column of the same dimensions (Pharmacia) equilibrated
with the above buffer supplemented with 0.5 M NaCl, and eluted
with the same.
Stimulation of Endothelial Cells
BPAECs were
plated on 6-well tissue culture plates in DM10F. After 5-7 days,
confluent monolayers were washed in PBS containing 1 mg/ml bovine serum
albumin and incubated with various agonists for the indicated time
intervals. Cells were lysed in ice-cold buffer containing 150 mM NaCl, 2 mM EDTA, 50 mM NaF, 1% Nonidet P-40,
0.1% sodium dodecyl sulfate, 10 µg/ml leupeptin, 10 µg/ml
aprotinin, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, and 20 mM Tris-HCl, pH 7.4. Lysates
were cleared by centrifugation at 14,000 G for 15 min, and
protein concentrations were determined using the DC Protein Assay
(Bio-Rad).
Immunoprecipitation of
Cell-surface lysyl residues of
confluent BPAECs were derivatized with 0.2 mg/ml sulfosuccinimidobiotin
(0.2 mg/ml) in PBS, pH 8.0, for 30 min at 4 °C. Monolayers were
washed in PBS and incubated at 4 °C for 1 h with either mAb LM609,
mAb P1F6, a polyclonal antiserum against ,
,
and
Integrins
integrins,
or a rabbit serum control. After detergent lysis and collection of
immune complexes on Protein A/Protein G-PLUS-agarose, detection of
immunoprecipitated proteins was performed following SDS-PAGE and
immunoblotting with streptavidin-horseradish peroxidase.
Immunoblotting and Immunoprecipitation of Tyrosine Kinase
Substrates
For detection of Vn, samples were electrophoresed
onto 7% SDS-polyacrylamide gels under reducing or non-reducing
conditions and transferred onto nitrocellulose. Vn was visualized
following addition of anti-Vn antiserum followed by alkaline
phosphatase-conjugated goat-anti rabbit IgG and development with
5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium.
Anti-phosphotyrosine immunoblotting was performed as described
previously(41) . In brief, following detergent lysis, equal
amounts of protein were electrophoresed onto 10% SDS-polyacrylamide
gels under reducing conditions. After electrophoretic transfer onto
nitrocellulose, phosphotyrosyl-containing proteins were detected using
an affinity purified anti-phosphotyrosine antibody that was previously
derivatized with sulfosuccinimidylbiotin, followed by addition of
streptavidin-horseradish peroxidase. For immunoprecipitation, cells
were lysed in a buffer containing 1% Nonidet P-40, incubated with
Protein A-agarose or Protein A/Protein G-agarose preadsorbed with the
appropriate antibodies, washed and subjected to SDS-PAGE, and
tranferred onto nitrocellulose. Detection of immunoprecipitated and/or
phosphotyrosyl-containing proteins was performed by adding the
appropriate antiserum/mAbs followed by horseradish
peroxidase-conjugated secondary antibodies. Blots were developed using
enhanced chemiluminescence.
Vitronectin and SC5b-9 Cause a Time- and
Concentration-dependent Increase in Protein Tyrosine Phosphorylation in
Bovine Pulmonary Artery Endothelial Cells
Addition of either
SC5b-9 or conformationally altered Vn to adherent BPAECs led to a
similar pattern of enhanced tyrosine phosphorylation of multiple
proteins that peaked at 3 min after the addition of agonist (Fig. 1). Proteins that demonstrated marked enhancement of
tyrosine phosphorylation corresponded to M values
of 28, 34, 38, 46, 52, 54, 69, and 125. At the lower concentrations
used, SC5b-9 was somewhat more effective at inducing enhanced protein
tyrosine phosphorylation than Vn; however, for both agonists, 400
µg/ml produced maximal enhancement of the accumulation of
phosphotyrosyl-containing proteins (Fig. 2). A similar
concentration-dependent increase in the extent of protein tyrosine
phosphorylation was seen using conformationally altered Vn further
purified by gel filtration (not shown).
Figure 1:
SC5b-9 and Vn induce a dynamic
enhancement of protein tyrosine phosphorylation in BPAECs. 600
µg/ml of either agonist was applied to confluent BPAEC monolayers
for the indicated time intervals at 37 °C, and cells were lysed and
subjected to SDS-PAGE and immunoblotting using anti-phosphotyrosine as
described under ``Experimental Procedures.'' A,
effect of SC5b-9; B, effect of purified
conformationally-altered Vn. Molecular weight markers appear at the
left.
Figure 2:
SC5b-9
and Vn induce a concentration-dependent enhancement of protein tyrosine
phosphorylation in BPAECs. The indicated concentrations of either
agonist was applied to confluent BPAEC monolayers for 3 min at 37
°C, and cells were lysed and subjected to SDS-PAGE and
immunoblotting using anti-phosphotyrosine as described under
``Experimental Procedures.'' A, effect of SC5b-9; B, effect of purified conformationally altered Vn. Molecular
weight markers appear at the left.
Multimeric Vn, but Not Monomeric Vn, Induces Enhanced
Protein Tyrosine Phosphorylation in Adherent BPAECs
Native Vn
obtained from two sources appeared as monomers on native SDS-PAGE (Fig. 3A, lane 1 and data not shown). Addition
of this monomeric Vn to adherent BPAECs did not induce a change in the
pattern or extent of protein tyrosine phosphorylation (Fig. 3B, lane 1). However, addition of
multimeric Vn, obtained by subjecting native Vn to treatment with urea,
which induces multimerization of individual Vn monomers(38) ,
led to a striking increase in the extent of protein tyrosine
phosphorylation (Fig. 3B, lane 2). A similar,
though less marked, increase in the extent of protein tyrosine
phosphorylation was observed in BPAECs treated with Vn purified by the
method of Yatohgo et al. ((40); Fig. 3B, lane
3). There was some variability in the extent of responsiveness of
BPAECs to different preparations Vn purified by this method, which may
have been due to their age and/or the duration of dialysis; these
variable have been shown to influence the extent of aggregation of Vn
monomers(42) . Consequently, we further fractionated purified
conformationally altered Vn by gel filtration, and obtained fractions
enriched in high molecular weight complexes of Vn, as assessed by
SDS-PAGE under native conditions and immunoblotting using anti-Vn
antiserum. Migration of Vn through gel filtration columns was somewhat
anomalous in that peaks of purified multimers appeared both in early
and very late fractions, which likely reflects ionic and/or hydrophobic
interactions between Vn and the bead matrix. We therefore used several
different gel filtration media, but obtained similar results (Fig. 3A, lanes 4, 5, and 7).
Regardless of the purification scheme, fractions enriched in multimers
of Vn were more potent per unit weight at inducing enhanced protein
tyrosine phosphorylation than unfractionated conformationally altered
Vn (compare lanes 4, 5, and 7 with lane 3 in Fig. 3B). To verify that BPAECs responded to the Vn
contained in both SC5b-9 and high molecular weight Vn complexes, rather
than to contaminating proteins, we depleted Vn from these samples using
saturating concentrations of anti-Vn antiserum, and applied the
resultant supernatants to BPAEC monolayers. Immunodepletion of Vn from
SC5b-9 or multimeric Vn completely blocked enhanced tyrosine
phosphorylation in BPAECs due to either agonist (Fig. 4),
confirming that Vn was responsible for eliciting this effect. Together,
these data indicate that Vn contained with either multimeric complexes
of Vn or SC5b-9 induces enhanced protein tyrosine phosphorylation in
adherent BPAECs.
Figure 3:
Effect of different preparations of Vn on
inducing enhanced protein tyrosine phosphorylation in BPAECs. A, equal amounts of Vn prepared as described under
``Experimental Procedures'' were subjected to SDS-PAGE under
native conditions and immunoblotted using anti-Vn antiserum. Lane
1, monomeric Vn; lane 2, multimeric Vn prepared by urea
treatment of monomeric Vn; lane 3, unfractionated
conformationally altered Vn; lane 4, late Bio-Gel P-100
fraction of conformationally-altered Vn; lane 5, early
Sephacryl S-300 fraction of conformationally altered Vn; lane
6, middle Sephacryl S-300 fraction of conformationally altered Vn; lane 7, late Sephacryl S-300 fraction of conformationally
altered Vn. B, Vn (400 µg/ml) derived from each of the
above fractions was added to confluent BPAEC monolayers at 37 °C
for 3 min. Cells were lysed and subjected to SDS-PAGE followed by
immunoblotting using anti-phosphotyrosine antibodies. C,
control monolayer incubated with PBS alone; lanes 1-7,
monolayers incubated with Vn preparations as described above in A. Molecular mass markers appear at
left.
Figure 4:
Vn
contained within SC5b-9 and high molecular weight complexes of Vn is
responsible for mediating enhanced protein tyrosine phosphorylation in
BPAECs. 600 µg/ml of either SC5b-9, unfractionated conformationally
altered Vn, or gel-purified high molecular weight Vn complexes were
applied to Protein A-agarose preadsorbed with either control non-immune
rabbit serum, or anti-Vn antiserum. Resulting supernatants were added
to confluent BPAEC monolayers for 3 min at 37 °C, and cells were
lysed and subjected to SDS-PAGE and immunoblotting using
anti-phosphotyrosine as described under ``Experimental
Procedures.'' Molecular weight markers appear at the
left.
RGD-containing Sequences within Multimeric Vitronectin
and SC5b-9, but Not Heparin-binding Domains, Are Responsible for
Mediating Enhanced Protein Tyrosine Phosphorylation
Several
epitopes within Vn have been implicated in binding to cell surface
receptors, including an RGD site (28) and a heparin-binding
domain(43, 44) . To assess whether either epitope
contributed to the promotion of enhanced protein tyrosine
phosphorylation, we incubated adherent BPAECs with either SC5b-9 or
multimeric Vn in the presence or absence of RGD or control
RGE-containing peptides, or heparin. Neither heparin, GRGDSP, or GRGESP
alone altered the pattern of protein tyrosine phosphorylation. However,
GRGDSP, but neither GRGESP nor heparin blocked the enhanced protein
tyrosine phosphorylation induced by either SC5b-9 or multimeric Vn (Fig. 5). Similar results were obtained when conformationally
altered Vn purified by the method of Yatohgo et al.(40) was used as a stimulus (not shown). Although this
experiment does not address which epitope(s) on Vn is responsible for
the binding of these Vn-containing ligands to BPAECs, it does show that
the RGD domain of Vn is necessary for inducing enhanced protein
tyrosine phosphorylation.
Figure 5:
The RGD
domain of Vn mediates enhanced protein tyrosine phosphorylation in
BPAECs. Confluent BPAECs were exposed to either 300 µg/ml GRGDSP (RGD) or GRGESP (RGE), 500 µg/ml heparin, 400
µg/ml SC5b-9 (S), or multimeric Vn (Vn) or
combinations of the above, as indicated, for 3 min at 37 °C. Cells
were lysed and subjected to SDS-PAGE and immunoblotting using
anti-phosphotyrosine as described under ``Experimental
Procedures.'' Molecular weight markers appear at the
left.
The
We first determined whether known Vn-binding
integrins were expressed at the cells' surfaces.
Immunoprecipitation of biotin-derivatized cell surface proteins by mAbs
against the Integrin Mediates
Enhanced Protein Tyrosine Phosphorylation Due to SC5b-9 and
Vitronectin
and
integrins revealed the presence of
these proteins, as assessed by the expected pattern of migration of
their subunits (45) following SDS-PAGE (Fig. 6). The
apparent faster mobility of the
subunit derived from
(compare two tops bands in Fig. 6) was similar to the findings of Smith et
al.(45) , and is of unknown significance. Although this
method does not provide an accurate assessment of the precise numbers
of integrins expressed on the cells' surfaces, their relatively
equivalent intensity of staining suggests a similar level of surface
expression. One or more
integrins were also expressed
at the surfaces of BPAECs (not shown), although we did not determine
whether
was one of these.
Figure 6:
Immunoprecipitation of surface-labeled
and
integrins derived from BPAECs. Two 9.6-cm
wells of
confluent BPAEC monolayers were incubated with sulfosuccinimidobiotin,
immunoprecipitated with mAbs against the indicated integrin, or with an
isotype-matched control, and subjected to SDS-PAGE and immunoblotting
with streptavidin-horseradish peroxidase as described under
``Experimental Procedures.'' Molecular weight markers appear
at the left.
To
identify which of these integrins is responsible for mediating enhanced
protein tyrosine phosphorylation due to soluble Vn-containing agonists,
we added antibodies that recognize these integrins to BPAECs during
addition of either SC5b-9 or Vn. Polyclonal and monoclonal antibodies
against completely blocked enhanced
protein tyrosine phosphorylation due to either agonist, whereas
anti-
antisera or anti-
mAbs did not (Fig. 7).
Figure 7:
Antibodies against the
integrin, but not against
or
integrins,
block enhanced protein tyrosine phosphorylation due to SC5b-9 and Vn in
BPAECs. Unstimulated BPAECs or BPAECs stimulated at 37 °C for 3 min
with 400 µg/ml of either SC5b-9 (A and C) or high
molecular weight Vn (B and D) were coincubated with
either mAb LM609 directed against
(20 µg/ml) or with mAb P1F6 directed against
(20 µg/ml) (A and B), or with a 1:50 dilution of either antiserum 838 against
or anti-
antiserum (C and D), and cells were lysed and subjected to
SDS-PAGE and immunoblotting using anti-phosphotyrosine as described
under ``Experimental Procedures.'' Molecular weight markers
appear at the left.
Clustering the
To further confirm that the
Integrin on Adherent BPAECs Mediates Enhanced Protein Tyrosine
Phosphorylation
integrin is capable of mediating
enhanced protein tyrosine phosphorylation in BPAECs, we incubated
adherent BPAECs with mAb LM609 followed by cross-linking of
surface-bound mAb with anti-mouse IgG. Clustering of
using mAb LM609 caused a marked
increase in protein tyrosine phosphorylation of multiple proteins in a
pattern similar to that seen after addition of SC5b-9 or Vn (compare Fig. 8with Fig. 1). Interestingly, the addition of either
mAb LM609 or polyclonal antiserum against
in the absence of a cross-linking antibody did not induce a
detectable increase in the extent of protein tyrosine phosphorylation (Fig. 7). Taken together, the above data suggest that clustering
of the
integrin mediates enhanced
protein tyrosine phosphorylation in BPAECs.
Figure 8:
Clustering of the
integrin is sufficient to trigger
enhanced protein tyrosine phosphorylation in BPAECs. Adherent BPAEC
monolayers were incubated at 4 °C for 30 min with 20 µg/ml of
either mAb LM609 or isotype-matched control mAb, washed, and further
incubated with 30 µg/ml donkey anti-mouse IgG at 37 °C for the
indicated time intervals, and cells were lysed and subjected to
SDS-PAGE and immunoblotting using anti-phosphotyrosine as described
under ``Experimental Procedures.'' Molecular weight markers
appear at the left.
The Tyrosine Kinase Substrates Paxillin, Cortactin,
Ezrin, Shc, and p125
To identify specific proteins that undergo
enhanced protein tyrosine phosphorylation in response to Vn, we
incubated adherent BPAECs with multimeric Vn, lysed the cells, and
performed immunoprecipitation using antisera against various tyrosine
kinase substrates followed by immunoblotting using
anti-phosphotyrosine. The cytoskeletal-associated proteins cortactin,
paxillin, and ezrin, the SH2 domain-containing protein Shc, and
p125, Undergo Enhanced Tyrosine
Phosphorylation in Bovine Pulmonary Artery Endothelial Cells in
Response to Vn
, underwent enhanced tyrosine phosphorylation in
response to Vn (Fig. 9). The enhanced tyrosine phosphorylation of
cortactin is similar to findings in brain microvascular endothelial
cells stimulated by activating surface-expressed ICAM-1(46) .
Figure 9:
Immunoprecipitation and
anti-phosphotyrosine immunoblotting of paxillin, cortactin, Shc, ezrin,
and p125 following addition of Vn in BPAECs. 400
µg/ml of multimeric Vn complexes were applied to confluent BPAEC
monolayers for 3 min at 37 °C, and cells were lysed and subjected
to immunoprecipitation followed by SDS-PAGE and immunoblotting using
anti-phosphotyrosine or the indicated antibodies as described under
``Experimental Procedures.'' Immunoblotting using antibodies
against the precipitated proteins was performed to compare extent of
recovery of immunoprecipitated proteins. Molecular weight markers
appear at the left.
integrins by multimeric Vn was required for this effect. 1)
Fractions enriched in Vn multimers were more potent than unfractionated
Vn in mediating enhanced protein tyrosine phosphorylation, whereas
monomeric Vn was incapable of triggering enhanced protein tyrosine
phosphorylation. 2) SC5b-9, a supramolecular complex which contains
several Vn molecules(47, 48) , was equally, if not more
potent in mediating protein tyrosine phosphorylation in these cells. 3)
A similar pattern of protein tyrosine phosphorylation was seen in
endothelial cell monolayers whose cell surface
was aggregated using mAb LM609
followed by goat anti-mouse IgG. Addition of mAb LM609 or
anti-
antiserum alone was ineffective
in altering the extent of protein tyrosine phosphorylation. 4) Addition
of mAb LM609 or polyclonal antiserum 838, directed against
, blocked enhanced tyrosine
phosphorylation mediated by either agonist. While these results are
consistent with the hypothesis that the valency of Vn is critical for
mediating enhanced protein tyrosine phosphorylation, we cannot discount
the possibility that receptor occupancy contributes to this response,
as well. However, in a recent study, clustering of
integrins in the absence of receptor occupancy was sufficient to
induce enhanced protein tyrosine phosphorylation in
fibroblasts(49) . Since a natural ligand for
has yet to be identified that
induces receptor clustering in the absence of receptor occupancy, the
importance of demonstrating a contributory role for receptor occupancy
in this response is questionable.
-mercaptoethanol (40) exposes a cryptic
heparin-binding domain within the Vn monomer, allowing its recognition
by several conformation-specific mAbs(42) , and predisposes Vn
molecules to multimerization(39, 42) . Stabilization of
the multimeric structure is mediated, in part, by disulfide bonding
since the resultant Vn multimers migrate as Vn monomers when applied to
SDS gels under reducing conditions(42) . Although this
conformationally altered Vn preparation is very different from native
Vn found in serum, its physical properties are shared by
substrate-bound and presumably ECM-derived Vn, as well as Vn bound to
the terminal complement complex ((SC5b-9)(42) ).
60% of the
binding to porcine aortic endothelial cells(44) . Second, we did
not assess binding of Vn to BPAECs. It is certainly possible that more
than one epitope within multimeric or aggregated Vn contributes to its
binding to endothelial cells of diverse origins. From our data,
however, we conclude that the RGD site within multimeric Vn is critical
for mediating enhanced protein tyrosine phosphorylation in BPAECs.
integrins are under a state of constant stimulation in
vivo. This raises the question of how these receptors signal
enhanced tyrosine phosphorylation in vivo, given the high
concentrations of Vn present in the plasma (0.1-0.5% of total
plasma protein; 50). In a recent study using different preparations of
Vn, Zanetti et al.(43) found that monomeric Vn bound
poorly, while aggregated Vn bound well, to endothelial cell monolayers.
In addition, our results suggest that native monovalent Vn is unlikely
to trigger enhanced protein tyrosine phosphorylation, even if a small
fraction of it were to bind to the endothelium in vivo. Situations in which a multivalent configuration of Vn is likely to
be present in the bloodstream include bacterial sepsis, which is
accompanied by the formation of SC5b-9(21) . During sepsis,
multivalent Vn complexes within SC5b-9 may bind tightly to luminal
receptors and initiate enhanced
tyrosine phosphorylation. This mode of signaling is reminiscent of at
least one other class of tyrosine kinase-mobilizing receptors, those
for the Fc portion of IgG (Fc
receptors). Despite the
presence of high circulating levels of monomeric IgG, these receptors
are optimally ligated by IgG aggregates or by IgG bound to surfaces,
such as bacterial cell walls (reviewed in Ref. 51). The analogy to
Fc
receptors is further underscored by the ability of
Vn to bind to a variety of Gram-positive and Gram-negative
bacteria(52) . It is possible that during bacteremia due to
these organisms, bacterial-bound Vn induces ligation and clustering of
endothelial
integrins, thus
promoting a highly localized stimulus for enhanced protein tyrosine
phosphorylation.
(
)The roles of
these particular tyrosine kinase substrates in effecting changes in the
cytoskeleton or contributing to the barrier function of endothelial
cell monolayers are unknown. It is particularly interesting that Shc,
an SH2 domain-containing ``adaptor'' protein implicated in
the Ras signaling pathway(61, 62, 63) ,
undergoes enhanced tyrosine phosphorylation by Vn-containing ligands.
Since Shc is a mediator of growth and differentiation in a variety of
cell types, and
is required for
angiogenesis in chorioallantoic membranes(26) , it is possible
that tyrosine phosphorylation mediated by this integrin is essential
for its angiogenesis-promoting capacity and that Shc plays a central
role in this process. In this respect, Shc may function in
integrin-mediated signaling pathways similar to its role in growth
factor receptor-mediated pathways. This is consistent with recent
studies demonstrating the association of a Shc-binding protein
implicated in Ras-mediated signaling, Grb2, with p125
in
fibronectin-stimulated fibroblasts (64) or with
in insulin-stimulated Rat-1
fibroblasts transfected with DNA encoding the human insulin receptor
(35).
using soluble
stimulii whose average size is large (720 nm
for
SC5b-9(48) ), compared with that of inter-endothelial junctional
pores (6.5-7.5 nm in size estimated from tracer
studies(65) ). Sieving properties of endothelial monolayers
grown on membrane filters show restricted diffusion of molecules whose
size is equal to, or greater than, fibrinogen (10.6 nm(66) ). It
is therefore unlikely that the Vn-containing ligands we used in this
study ligated
integrins located on
the abluminal surface of the endothelium, at sites of focal adhesion.
Since p125
is concentrated in areas of focal adhesion to
the underlying substrate(13, 16) , it is possible that
there is considerable spatial separation between ligated
receptors and phosphorylated
p125
molecules. Alternatively, a small fraction of
p125
may be freely diffusible in the cytosol or
associated with the subset of
receptors that are accessible to soluble Vn.
may mediate changes in
endothelial cell monolayer permeability to macromolecules in
vitro, similar to results we have obtained in isolated lung
venules.
In contrast, a previous study examining
endothelial cell monolayer barrier function in human umbilical vein
endothelial cells reported that antibodies against the
integrin, but not against
integrins, increased transendothelial flux of
macromolecules(67) . Since the expression and location of
is very likely dependent upon cell
type and culture conditions, and the authors showed a relative paucity
of staining for
subunits at intercellular borders,
their inability to demonstrate
-mediated changes in monolayer
permeability to macromolecules in these cells is not surprising. In any
case, the relationship between
-mediated enhanced protein tyrosine
phosphorylation and endothelial cell barrier function awaits further
study.
], cytosolic-free calcium concentration;
DM10F, Dulbecco's modified Eagle's medium supplemented with
10% fetal bovine serum; PBS, phosphate-buffered saline; Vn,
vitronectin; mAb, monoclonal antibody; PAGE, polyacrylamide gel
electrophoresis.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.