Division of Cardiovascular Research, Research Institute, The Hospital for Sick Children and Departments of Pediatrics, Pathology, and Medicine, University of Toronto, Toronto, Ontario, Canada M5G 1X8
Tenascin-C (TN-C) is induced in pulmonary
vascular disease, where it colocalizes with proliferating
smooth muscle cells (SMCs) and epidermal growth factor (EGF). Furthermore, cultured SMCs require TN-C
for EGF-dependent growth on type I collagen. In this study, we explore the regulation and function of TN-C
in SMCs. We show that a matix metalloproteinase
(MMP) inhibitor (GM6001) suppresses SMC TN-C
expression on native collagen, whereas denatured collagen promotes TN-C expression in a 3 integrin-
dependent manner, independent of MMPs. Floating
type I collagen gel also suppresses SMC MMP activity
and TN-C protein synthesis and induces apoptosis, in
the presence of EGF. Addition of exogenous TN-C to
SMCs on floating collagen, or to SMCs treated with
GM6001, restores the EGF growth response and "rescues" cells from apoptosis. The mechanism by which
TN-C facilitates EGF-dependent survival and growth
was then investigated. We show that TN-C interactions with
v
3 integrins modify SMC shape, and EGF-
dependent growth. These features are associated with
redistribution of filamentous actin to focal adhesion
complexes, which colocalize with clusters of EGF-Rs,
tyrosine-phosphorylated proteins, and increased activation of EGF-Rs after addition of EGF. Cross-linking
SMC
3 integrins replicates the effect of TN-C on
EGF-R clustering and tyrosine phosphorylation. Together, these studies represent a functional paradigm
for ECM-dependent cell survival whereby MMPs upregulate TN-C by generating
3 integrin ligands in type
I collagen. In turn,
v
3 interactions with TN-C alter
SMC shape and increase EGF-R clustering and EGF-dependent growth. Conversely, suppression of MMPs
downregulates TN-C and induces apoptosis.
TENASCIN-C (TN-C)1 is an extracellular matrix
(ECM) glycoprotein that is prominent in embryonic
and adult tissues that are actively remodeling (Chiquet-Ehrismann et al., 1995 A functional role for TN-C in modulating vascular
smooth muscle cell (SMC) proliferation has been suggested by studies demonstrating increased TN-C synthesis
after wounding injury in vivo (Hedin et al., 1991 Although the mechanism by which TN-C cooperates
with soluble growth factors to generate a mitogenic response in normal SMCs is not understood, recent studies
with other cell types show that integrin and growth factor
receptor signaling components may accumulate on the actin-based cytoskeletal scaffold to form a specialized focal
adhesion complex (Miyamato et al., 1995a In this study, we first investigated the relationships between MMPs, TN-C, and EGF-dependent SMC survival.
Using SMCs cultured on attached and floating collagen
gels as a model system, we show that net MMP activity,
TN-C expression, and SMC survival are suppressed on
floating collagen gels. Furthermore, we demonstrate that
Cell Culture
Vascular SMCs were isolated from pulmonary arteries of 300-g adult male
Sprague-Dawley rats. Briefly, arteries were harvested from anaesthetized
animals and placed in cold sterile PBS, pH 7.6. Endothelium was removed
by gently scraping the lumenal surface with a scalpel blade, and the adventitia was also removed from the vessel. The medial layer was minced using
scalpel blades and incubated at 37°C in medium 199 (M199; GIBCO BRL,
Gaithersburg, MD) supplemented with 0.1% collagenase I (Sigma Chemical Co., St. Louis, MO.), and 0.1% BSA (Boehringer-Mannheim, Laval
Canada) for 1 h with gentle rotation. Tissue was collected by centrifugation at 300 g, resuspended in M199/collagenase I solution, and incubated overnight at 37°C with gentle rotation. Smooth muscle cells were harvested by
centrifugation and were routinely maintained in M199 containing 10%
heat-inactivated FBS (Intergen, Purchase, NY), 10 U/ml penicillin G sodium, 10 mg/ml streptomycin sulfate, 0.25 mg/ml amphotericin B, and 0.1 mg/ml gentamicin sulfate (GIBCO BRL). Cells were passaged by
trypsinization using 0.05% trypsin/EDTA (GIBCO BRL). Vascular
SMCs were identified by their characteristic hills-and-valley morphology
and immunohistochemical staining for Collagen gels were prepared based on methods that have been previously described (Jones and Rabinovitch., 1996). Briefly, 0.8 ml of a 3.1 mg/
ml solution of bovine dermal type I collagen (Vitrogen 100; Collagen
Corp., CA), 0.1 ml of 0.1 M NaOH, and 0.1 ml of 10× PBS were mixed at
4°C for a final collagen concentration of 2.48 mg/ml. To determine the effect of TN-C on SMC behavior, neutralized collagen was supplemented
with 15 µg/ml of human TN-C protein (Chemicon International, Temecula, CA). Fibrillogenesis was initiated overnight in a humid 5% CO2 environment at 37°C, and the gels were rinsed extensively (three times for 3 h)
with M199 containing 0.1% BSA before use. Confluent cultures of rat PA
SMCs were serum starved in M199/0.1% BSA for 48 h. Smooth muscle cells were collected by trypsinization and centrifugation, and cell number
was determined using an improved Neubauer hemocytometer (American
Optical, Buffalo, NY). Cell pellets were resuspended in M199/0.1% BSA
plus 0.5% serum, and a 1-ml aliquot of SMCs was seeded onto the surface
of each gel. 6 h later, cells were rinsed in M199 and were thereafter cultured in M199/0.1% BSA.
Smooth Muscle Growth on Collagen Gels
We used collagen gels and heat-denatured collagen to determine the interrelationships between TN-C, MMP activity, Detection of Matrix Metalloproteinases
Conditioned medium was collected from cells maintained on attached or
floating collagen gels for 4, 8, 12, and 24 h in M199 with 50 ng/ml EGF. For
each culture and at every time point, SMCs were rinsed gently three times
with culture medium and then reincubated with 1 ml of fresh medium. At
the designated time points, conditioned medium was harvested from the
cultures, and equal volumes of medium were lyophilized overnight. Conditioned medium was mixed 3:1 (vol/vol) with 4× sample buffer (10%
SDS, 25% glycerol in 150 mM Tris-Cl, pH 6.8) and separated on nonreducing 10% polyacrylamide gels that contained 0.1% gelatin (Sigma
Chemical Co.). After electrophoresis, gels were soaked in 2.5% Triton
X-100 to remove SDS and incubated in substrate buffer (50 mM Tris-Cl,
pH 7.5, 5 mM CaCl2) for 18 h at 37°C. The gels were then stained with
Coomassie blue R250 (Bio-Rad Labs, Hercules, CA). Gelatinases appear
as a clear zone on a blue background. To demonstrate the ability of
GM6001 to inhibit MMPs, we incubated gelatin zymograms in substrate
buffer containing 2 µM of this inhibitor. This treatment abolished MMP activity. For detection of the pro-form of MMP-2, 20 µg of total protein
contained in conditioned medium harvested from SMCs cultured on attached and floating collagen for 24 h in serum-free medium (SFM) was
subjected to SDS-PAGE electrophoresis. Proteins were electrophoretically transferred to nylon membranes, blocked for 1 h at 37°C in wash
buffer (10 mM Tris, pH 7.5, 10 mM NaCl, and 0.1% Tween-20), containing 0.1% BSA and 1% normal goat serum (Sigma Chemical Co.), and
then with an anti-MMP-2 rabbit polyclonal antisera (a kind gift from Drs.
M. Silverman and M. Ailenburg, Department of Medicine, University of
Toronto, Toronto, Canada) diluted 1:200 in wash buffer for 30 min at
37°C. To detect MMP-2, membranes were then incubated with a horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (GIBCO
BRL) diluted 1:3,000 in wash buffer, rinsed in wash buffer three times for
15 min, visualized by enhanced chemiluminescence (ECL kit; Amershan
Corp., Arlington Heights, IL), and exposed to film (X-Omat; Kodak,
Rochester, NY).
Immunofluorescent Detection of
Phosphorylated Proteins, Actin, Epidermal Growth
Factor Receptors, and Vinculin
For immunofluorescent studies, serum starved SMCs were cultured on
glass coverslips (12 mm, No.1 thickness) coated with 2.48 mg/ml of neutralized type I collagen (+/ Biochemical Analysis of
Tyrosine-phosphorylated Proteins, Tenascin-C, and
Epidermal Growth Factor Receptors
Tyrosine-phosphorylated proteins were analyzed by immunoblotting
whole cell lysates, SMC membrane-enriched protein fractions, or EGF-R
immunoprecipitates with a horseradish peroxidase-conjugated antiphosphotyrosine (RC20H; Transduction Labs, Lexington, KY). 4 × 106 SMCs
cultured for 24 h on 10-cm-diam collagen gels (+/ To enrich for membrane proteins, SMCs were harvested at the appropriate time points in ice-cold buffer containing 50 mM Hepes, 1 mM benzamidine, 1 mM PMSF, 1 µg/ml aprotinin, 5 µg/ml leupeptin, 2 mM NaF, 2 mM Na3VO4, 1 mM MgCl2, and 0.25 M sucrose, pH 7.4 at 4°C. Cells were
homogenized with a Dounce B glass homogenizer (60 strokes), and intact
cells and nuclei were sedimented by centrifugation at 3,000 g for 5 min at
4°C. The resulting supernatants were centrifuged at 100,000 g for 1 h at
4°C, and the membrane-enriched pellet was solubilized by sonication in
homogenization buffer supplemented with 1% Triton X-100. Protein content was determined using the Bio-Rad protein assay according to the
manufacturer's instructions. For EGF-R immunoprecipitation experiments, 15 µg of membrane-enriched protein, resuspended in 0.5 ml RIPA
lysis buffer (25 mM Tris, pH 7.2, 0.1% SDS, 1% Triton X-100, 1% sodium
deoxycholate, 150 mM NaCl, and 1 mM EDTA) containing 1 mM benzamidine, 1 mM PMSF, 1 µg/ml aprotinin, 5 µg/ml leupeptin, 2 mM NaF,
and 2 mM Na3VO4 was precleared with normal rabbit IgG and protein
A-Sepharose (Sigma Chemical Co.) for 1 h at 4°C. Supernatants were
then incubated for 1 h at 4°C with the rabbit polyclonal anti-EGF-R antibody diluted 1:400 in RIPA buffer and precipitated with protein A-Sepharose. Pellets were washed three times with ice-cold RIPA buffer and once in 50 mM Tris, pH 7.4. Membrane fractions (15 µg) and EGF-R immunoprecipitates were boiled in reducing sample buffer containing 0.2 M Tris-HCl,
pH 8.8, 18% glycerol, 4% SDS, 0.01% bromophenol blue, and 10%
To cluster In these studies, we also used immunofluorescence to demonstrate the
clustering of Immunoprecipitation of Epidermal Growth Factor
Receptors and Tenascin-C Protein
For immunoprecipitation of radiolabeled EGF-R and TN-C proteins, 2.5 × 104 serum-starved SMCs were cultured for 24 h on collagen gels in 35-mm-diam tissue culture dishes. To determine the effect of TN-C on EGF-R
synthesis, collagen gels were supplemented with 15 µg/ml of TN-C. 24 h
after plating, SMCs were rinsed twice and incubated in cysteine/methionine-free medium (ICN Biomedicals, Inc.) supplemented with 0.1% BSA
for 3 h and then pulse-labeled with 100 µCi/ml [35S]methionine/cysteine
translabel (ICN Biomedicals, Inc.) for 18 h. To determine the effects of
MMP inhibition on TN-C protein synthesis, cells were treated with 1- or
2-µm GM6001 in DMSO or with DMSO alone. Tissue culture medium
was collected in RIPA buffer and cleared by centrifugation. Total counts
per minute were normalized by TCA precipitation and equal counts per
minute (5 × 105 cpm/sample) were immunoprecipitated with 5 µl rabbit
polyclonal antisera raised against rat EGF-Rs (RK-2) or human TN-C
(GIBCO BRL). Immune complexes were precipitated with 25 µl protein
A-Sepharose (Sigma Chemical Co.), washed in 1× RIPA buffer, 50 mM
Tris, pH 7.4, and resuspended in 40 µl 2× SDS-PAGE sample buffer. Proteins were separated on 6% SDS-polyacrylamide gels, fixed, dried, and exposed to film (X-Omat; Kodak) for 3 d.
Apoptosis Assay
Internucleosomal DNA fragmentation is a characteristic of apoptosis and
was used to assess programmed cell death in SMCs cultured on attached
and floating type I collagen gels (+/ Statistical Analyses
All statistical assessments were compared by one-way analysis of variance
and Student Newman Keuls post hoc analysis. A P value of <0.05 was
considered statistically significant. Mean values ±SEM are given in the
figures.
Matrix Metalloproteinases and Tenascin-C
Are Coordinately Expressed in Vascular Smooth
Muscle Cells
When chick embryo fibroblasts are cultured on attached
type I collagen gels, they express TN-C protein, whereas
culture on floating collagen leads to suppression of TN-C
expression (Chiquet-Ehrismann et al., 1994
The production and activity of MMPs under these conditions was then examined by gelatin substrate zymography. Two proteins with approximate molecular masses of
68 and 57 kD were detected in conditioned medium harvested from SMCs cultured on attached and floating collagen (Fig. 1 C). Densitometric analysis of these gelatinases showed that, relative to attached collagen, the
activities of the 68- and 57-kD gelatinase decreased with
time on floating collagen (Fig. 1 D). Under the denaturing
and nonreducing conditions used for zymography, the pro-form of MMP-2 has an apparent molecular mass of 68 kD,
whereas the active form appears at ~57 kD (Ailenburg et
al., 1993 Matrix Metalloproteinases and Previous studies have shown that interstitial collagenases,
including MMP-2, are capable of degrading fibrillar native
type I collagen to generate collagen fragments (Aimes and
Quigley, 1995 To first establish whether MMPs regulate TN-C expression, we cultured SMCs on attached type I collagen gels in
the presence of a specific MMP inhibitor, GM6001 (Galardy,
1993
We next assessed TN-C protein expression on native
versus heat-denatured collagen. Western immunoblot analysis showed that TN-C protein expression is upregulated on
denatured collagen (Fig. 2 B), and that this substrate obviated the need for MMPs (Fig. 2 C). To address whether
TN-C protein expression is regulated by Matrix Metalloproteinases and Tenascin-C Promote
Smooth Muscle Cell Survival
Since MMPs regulate SMC TN-C protein synthesis on native type I collagen, and exogenous TN-C cooperates with
EGF to facilitate SMC growth on this substrate (Jones and
Rabinovitch, 1996
Tenascin-C Collaborates with the Tenascin-C has the capacity to bind and interact with multiple cell surface receptors, including the
Tenascin-C Promotes Accumulation of
F-actin, Tyrosine-phosphorylated Proteins, and
Epidermal Growth Factor Receptors to
the Focal Adhesion Complex
The effects of TN-C-
Given that high-affinity EGF-Rs bind directly to actin
(den Hartigh et al., 1992 Tyrosine Phosphorylation of Epidermal Growth Factor
Receptors Is Enhanced by Exogenous Tenascin-C
Having established that culturing SMCs on exogenous
TN-C promotes EGF-R clustering, we next determined
the effects of TN-C on EGF-R protein synthesis and tyrosine phosphorylation of this receptor. Immunoprecipitation of radiolabeled whole cell lysates with an anti-EGF-R
antibody showed that the levels of receptor protein synthesis were identical on collagen and TN-C-supplemented gels (Fig. 6 A). Consistent with our immunofluorescent
studies, Western immunoblotting of membrane-enriched
fractions with an antiphosphotyrosine antibody revealed
that there was increased tyrosine phosphorylation in the
presence of exogenous TN-C (Fig. 6 B). In addition, on
both type I collagen and TN-C-supplemented cultures,
EGF treatment resulted in a transient increase in protein tyrosine phosphorylation that was enhanced by TN-C. Of
particular interest was a 170-kD tyrosine-phosphorylated
species that corresponds in molecular mass to the EGF-R.
Also noted was an increase in an ~125-kD species, which
may represent focal adhesion kinase (Fig. 6 B). To specifically determine whether TN-C promotes EGF-R activation, EGF-Rs were immunoprecipitated from SMC membrane fractions, and their activation status was assessed
using Western immunoblotting with an antiphosphotyrosine antibody. This confirmed that EGF-R phosphorylation occurs by 10 min after addition of EGF, and that this
response is enhanced in TN-C-supplemented as opposed
to SMC cultures on collagen gels alone (Fig. 6 C).
We next examined the effects of blocking TN-C-
To determine whether clustering of
To further assess the effect of A number of studies have described the induction of TN-C
in remodeling vascular tissues, where it has been functionally linked to SMC proliferation (Hedin et al., 1991
Recent studies by Chiquet-Ehrismann et al. (1994) It is well established that specific cell-ECM interactions
provide critical cues that regulate proliferation, migration,
differentiation, and cell viability (Jones et al., 1993 Matrix metalloproteinase-mediated alterations in cell-
ECM interactions have profound effects on receptor-mediated intracellular signaling and subsequent cell behavior
(for review see Bausbaum and Werb, 1996 Considerable evidence demonstrates that regulation of
cell shape and tissue organization by the ECM profoundly
influences gene expression, differentiation, and growth behavior (Folkman and Moscona, 1978 Our studies comparing SMC growth and survival on attached and floating collagen gels add to previous work
that suggests that mechanical factors are able to modulate
growth factor-dependent functions, by relating this property to alterations in TN-C expression, and concomitant
changes in cell shape and growth factor receptor activation. For example, fibroblasts in mechanically stressed collagen continue to proliferate in response to growth factors (Nakagawa et al., 1989 Binding of EGF to its receptor leads to dimerization,
which is essential to the subsequent activation of intrinsic
tyrosine kinase activity (Schlessinger, 1988 Most integrins do not possess intrinsic kinase activity,
and their ability to generate a signal after ligation to ECM
appears to be dependent upon the formation of specialized structures comprised of multiple components, including clustered integrins, actin, growth factor receptor signaling molecules, and cytoskeletal-associated proteins
such as vinculin and focal adhesion kinase (Miyamoto et
al., 1995a; Plopper et al., 1995 In conclusion, our results represent a model for neointimal formation, whereby induction of MMPs upregulates
TN-C expression, which in turn facilitates growth factor-
dependent SMC proliferation. Based on the present studies,
and our work that has linked increased TN-C expression
and SMC proliferation in vivo (Jones and Rabinovitch, 1996), where it is often coexpressed
with matrix metalloproteinases (MMPs), a family of zinc-dependent ECM-degrading enzymes (Hedin et al., 1991
;
Talhouk et al., 1991
; Jones et al., 1995
; Latijnhouwers et al.,
1996
; Vaalamo et al., 1996
). For example, during postlactational involution of the mammary gland, increased levels
of TN-C and stromelysin-1 directly contribute to the loss
of tissue-specific gene expression (Sympson et al., 1994
;
Jones et al., 1995
) and the onset of apoptosis (Boudreau et
al., 1995a
, 1996
), which together characterize this stage of
development (Strange et al., 1992
). In mature blood vessels subjected to experimental balloon catheter injury, TN-C
and MMP-2 expression are induced before the development of occlusive neointimal lesions (Mackie et al., 1992
;
Bendeck et al., 1994
; Hedin et al., 1991
; Zempo et al., 1994
;
Strauss et al., 1996
), indicating that these proteins may also
share a common function during vascular remodeling. In
addition, since TN-C and MMP-2 each have the capacity
to bind the
v
3 integrin (Prieto et al., 1993
; Sriramarao et
al., 1993
; Brooks et al., 1996
) and their expression is sensitive to treatments that alter the integrity of the actin cytoskeleton (Aggeler et al., 1984
; Chiquet-Ehrismann et al.,
1994
), it is possible that these proteins are regulated in an
interdependent manner. Indeed, studies by Tremble et al.
(1994)
have demonstrated that TN-C-supplemented fibronectin matrices stimulate MMP expression in cultured
rabbit synovial fibroblasts. Evidence that MMPs may regulate TN-C in a reciprocal manner, however, has not been
forthcoming.
), or by
treatment with vasoactive peptides and growth factors in
tissue culture (Mackie et al., 1992
; Sharifi et al., 1992
). Our
recent studies in rat and human hypertensive pulmonary arteries (Jones and Rabinovitch, 1996
; Jones et al., 1997
),
as well as those in vessels from spontaneously hypertensive rats and human vein grafts (Hahn et al., 1995
; Chen et
al., 1996
), have shown that TN-C colocalizes with proliferating SMCs, as well as EGF (Jones et al., 1997
). An additional link between TN-C expression and growth factor-
dependent SMC proliferation was also provided by tissue
culture studies that demonstrated that TN-C potentiates bFGF-dependent SMC growth and is critical for EGF-dependent SMC growth on type I collagen (Jones and
Rabinovitch, 1996
). Similarly, End et al. (1992)
reported
that TN-C collaborates with EGF to promote growth of
several cell types, including fibroblasts and SMCs isolated
from spontaneously hypertensive rats.
,b; Plopper et
al., 1995
), where they collaborate to regulate activation of
mitogen-activated protein kinases and receptor tyrosine
kinases, including the EGF receptor (EGF-R) (Miyamoto
et al., 1996). Mechanistically, epidermal growth factor receptors have been shown to directly interact with actin
(den Hartigh et al., 1992
), and from a functional standpoint, this association greatly enhances activation of the
EGF-R and its downstream substrates (Diakonova et al.,
1995
; Gronowski and Bertics, 1995
). Since TN-C has been
shown to alter the integrity of the actin cytoskeleton (Murphy-Ullrich et al., 1991
; Chung et al., 1996
), it is therefore
possible that this ECM component modulates EGF-R signaling through its effects on the actin cytoskeleton and associated focal adhesion components.
3 integrins support basal levels of TN-C expression on attached native type I collagen and that a specific inhibitor
of MMPs (GM6001) suppresses this expression and limits
cell survival. Moreover, addition of exogenous TN-C to
SMC cultures in which MMP activity and TN-C production have been suppressed supports SMC survival and
EGF-dependent growth. Culturing SMCs on denatured
collagen obviates the requirement for MMPs and directly
promotes TN-C expression in a
3 integrin-dependent
manner. We next explored the mechanism by which TN-C
facilitates this effect, and we show that TN-C binding to
the
v
3 integrin supports EGF-dependent SMC growth,
and that this interaction leads to changes in cell morphology underscored by the reorganization of the actin cytoskeleton. This structural alteration is accompanied by
clustering of EGF-Rs and accumulation of tyrosine-phosphorylated proteins at the cell periphery within focal adhesion complexes. Addition of EGF to these physically and biochemically primed cells leads to increased SMC
EGF-R activation and growth. Together, these data provide a functional paradigm for the regulation of TN-C by
MMPs in actively restructuring tissues and for how this
ECM component interacts with
3 integrins to promote
EGF-dependent growth and survival.
MATERIALS AND METHODS
-smooth muscle actin.
3 integrins, and EGF-dependent SMC survival and growth. For these experiments, 2 × 104 cells were
plated on collagen gels as described above. For anti-
v
3 antibody blocking studies, SMCs were plated on collagen gels in the presence of 15 µg/ml
LM609 monoclonal antibody (a kind gift from Dr. David Cheresh, Scripps
Research Institute, La Jolla, CA) or with 15 µg/ml of control IgG (DAKO
Corp., Carpenteria, CA). For
3 integrin studies on native and denatured
collagen, SMCs were plated in the presence of 25 µg/ml anti-
3 monoclonal antibody (PharMingen, San Diego, CA) or with 25 µg/ml of control IgG (DAKO Corp.). For MMP inhibitor studies, 1-2 µm GM6001 (a kind gift
from Dr. Stu Sweidler, Glycomed, Alameda, CA) in DMSO, or an equivalent
volume of DMSO alone, was added to cultures 24 h after plating. GM6001
(N- [2 (R) - 2 - ( hydroxamidocarbonylmethyl ) - 4 - methylpentanoyl] - L - tryptophane methylamide) is a noncytotoxic synthetic inhibitor that is specific
for MMPs. The hydroxamic group of GM6001 binds to the critical active
site zinc atom present in MMPs. In addition, the isobutyl group and tryptophan side chain of GM6001 also binds to subsites on MMPs, which normally bind side chains of ECM proteins (Galardy, 1993
; Boudreau et al., 1995a
; Strauss et al., 1996
). For assessment of the effects of heat denatured
collagen, cells were plated on 50 µg of type I collagen/35-mm-diam dish
that had been boiled for 30 min with 0.02 M acetic acid, and then air dried
to the bottom of each dish. Before plating cells, collagen substrates were
rinsed extensively, (three times for 1 h) with M199/0.1% BSA. To "float"
the collagen gels, we detached them from the plastic substratum 24 h after plating using a spatula. 24 h after plating, cells were cultured in M199/
0.1% BSA either with or without 50 ng/ml EGF (GIBCO BRL). The
number of cells retained on collagen gels was determined 72 h after plating. The dose of EGF chosen to be stimulatory to SMC growth was determined in pilot studies and previously published by our group (Jones and
Rabinovitch, 1996
). All experiments were performed in triplicate. Attachment efficiencies were determined by counting the number of cells in the
medium using a hemocytometer or Coulter counter. The number of attached cells (number of cells seeded
number of cells in the medium)
was expressed as a percentage of the number of cells seeded. To assess the
number of cells retained on collagen, gels were digested with 1 mg/ml collagenase type II (Sigma Chemical Co.) for 1 h at 37°C. Free cells were pelleted by centrifugation at 4°C for 10 min at 300 g and suspended in PBS
containing 0.05% trypsin, and cell number was determined by counting aliquots in triplicate using a hemocytometer.
15 µg/ml TN) in M199/0.1% BSA. For actin,
EGF-Rs, and tyrosine-phosphorylated proteins, cells were exposed to 50 ng/ml EGF in M199/0.1% BSA for 30 min or subjected to control treatment with M199/0.1% BSA alone. To determine the effects of blocking
the
3 integrin on EGF-R distribution, SMCs were preincubated with 25 µg/ml anti-rat
3 integrin monoclonal antibody (CD61; PharMingen) before plating on collagen- and TN-C-enriched substrates. At the appropriate time point, cells were rinsed in PBS and then fixed. For detection of
F-actin, SMCs were fixed with extraction buffer (60 mM Pipes, 25 mM
Hepes, 10 mM EDTA, 2 mM MgCl2, pH 6.9) supplemented with 0.2%
Triton X-100 for 5 min at 4°C. Smooth muscle cells were then washed
three more times for 5 min in extraction buffer without Triton X-100.
Fixed cells were incubated with 2.5 U of rhodamine-phalloidin (Molecular
Probes, OR) in PBS for 20 min at 37°C before rinsing briefly with PBS. To
detect EGF-Rs, tyrosine-phosphorylated proteins, and vinculin, SMCs
were fixed in 2% paraformaldehyde (Sigma Chemical Co.) for 10 min at
room temperature and rinsed in PBS containing 0.1 M glycine for 30 min.
For tyrosine-phosphorylated proteins and vinculin, SMCs were permeabilized with 0.2% Triton X-100 in PBS for 5 min and washed three more
times. The fixed cells were incubated for 1 h at 37°C in wash buffer (PBS/
1% BSA), supplemented with 10% normal goat serum for EGF-Rs, and
then incubated for 1 h at 37°C with anti-EGF-R antisera (RK-2, diluted
1:100; a kind gift from Dr. Irit Lax, New York University Medical Center,
New York), antiphosphotyrosine monoclonal antibody (clone 4G10; 5 µg/
ml; Upstate Biotechnology Inc., Lake Placid, NY), antivinculin monoclonal antibody (diluted 1:50; Sigma Chemical Co.), or appropriate preimmune sera and IgG controls in wash buffer. After being washed three
times, coverslips were incubated for 1 h at 37°C either with fluorescein-conjugated goat anti-rabbit antibody (diluted 1:100; Sigma Chemical Co.) or with goat anti-mouse antibody (diluted 1:100; Sigma Chemical Co.).
For double immunofluorescent staining experiments, all cells were fixed
in 2% paraformaldehyde. For vinculin and EGF-Rs, cells were permeabilized in methanol for 2 min and were then air dried for 1 h at room temperature. For vinculin, tyrosine-phosphorylated proteins, and F-actin, fixed
cells were washed in PBS containing 0.2% Triton X-100. All cells were
then washed with PBS/0.1 M glycine and were sequentially incubated with
appropriate antisera, antibodies, or rhodamine-phalloidin as described
above. After washing, all coverslips were mounted onto glass slides using
Antifade reagent (Molecular Probes). Observations and photomicrographs were obtained with a fluorescent microscope (Olympus Corp.,
Lake Success, NY) using epifluorescence.
TN-C) were treated
with EGF (50 ng/ml) for 10 and 30 min or were further incubated in M199/
0.1% BSA alone. Tenascin-C protein expression on collagen gels was assessed by immunoblotting equal numbers (2 × 104) of cell lysates with an
anti-chicken TN-C rabbit polyclonal antisera (pK7; gift from Dr. M. Schachner, Swiss Federal Institute of Technology, Zurich, Switzerland).
To determine the effect of the
3 integrin on TN-C-dependent EGF-R
phosphorylation, SMCs were cultured on TN-C-supplemented collagen
gels with 25 µg/ml of anti-rat
3 integrin monoclonal antibody (CD61;
PharMingen) or with control IgG (DAKO Corp.).
-mercaptoethanol. All samples were separated in 7% SDS-polyacrylamide gels. Proteins were transferred to Immobilon polyvinyl fluoride
membranes (Millipore Corp., Milford, MA), and these were blocked for
1 h at 37°C in wash buffer (10 mM Tris, pH 7.5, 10 mM NaCl, and 0.1%
Tween-20), supplemented with 0.1% BSA. To detect tyrosine-phosphorylated proteins, blots were incubated for 30 min with horseradish peroxidase-conjugated antiphosphotyrosine antibody, diluted 1:2,500 in wash
buffer. To detect TN-C protein, membranes were sequentially incubated
with anti-TN-C antisera and a horseradish peroxidase-conjugated goat
anti-rabbit secondary antibody (GIBCO BRL) diluted 1:5,000 in wash
buffer. Thereafter, all membranes were rinsed in wash buffer three times
for 15 min, and tyrosine-phosphorylated and TN-C proteins were visualized by enhanced chemiluminescence (Amersham ECL kit) before exposure to film (X-Omat; Kodak).
3 Integrin Cross-Linking Studies
3 integrins, SMCs were first cultured on collagen gels and were
then washed gently with medium and incubated with 150 µg/ml anti-
3
monoclonal antibody (PharMingen) diluted in M199/0.1% BSA for 60 min. The SMCs were then rinsed with medium and incubated either with
20 µg/ml of goat anti-mouse IgG F(ab
)2 fragment (ICN Biomedicals,
Inc., Costa Mesa, CA) or with medium alone for 60 min at 37°C. For tyrosine-phosphorylation studies, SMCs were rinsed in medium and treated
with 50 ng/ml EGF for 10 or 30 min. Control cultures were maintained in
M199/0.1% BSA. Epidermal growth factor receptor immunoprecipitates
were analyzed by immunoblotting with antiphosphotyrosine antibodies as
described above.
3 integrins. Accordingly, SMCs were rinsed in PBS and
fixed in acetone at
20°C for 1 min. Cells were rehydrated in PBS,
blocked in 1% PBSA for 1 h at 37°C, and incubated with a fluorescein-conjugated goat anti-mouse antibody diluted 1:100 in 1% PBSA for 30 min at 37°C, before being washed and visualized by epifluorescence. In
these experiments, EGF-R distribution was also examined by immunofluorescence as described above.
TN-C). 48 h after floating, cells
were rinsed in PBS and incubated on ice with DNA lysis buffer (10 mM
Tris, pH 8, 100 mM NaCl, 2 mM EDTA, and 0.5% SDS) for 5 min with rotation. Lysates were incubated with 1 µg/ml proteinase-K (Boehringer-Mannheim Corp.) at 37°C for 24 h with rotation. Genomic DNA was purified by sequential extraction with phenol/chloroform and was precipitated
overnight in ethanol at
20°C. DNA pellets were treated with 10 µg/ml
RNase A for 1 h at room temperature, and DNA was then quantified
based on optical density. 10 µg of genomic DNA was analyzed by electrophoresis through 1.2% agarose gels and visualized with ethidium bromide.
RESULTS
). Using this
culture method as a model system, we first compared SMC
morphology, TN-C production, and MMP activity and expression on attached and floating collagen gels. Within the
first few hours of releasing attached collagen gels into the culture medium, SMCs lost their elongated morphology
and became rounded (Fig. 1 A). Immunoprecipitation of
radiolabeled TN-C protein from SMC conditioned medium after 24 h (Fig. 1 B) showed that synthesis of two
TN-C protein isoforms, of apparent molecular masses 220 and 180 kD, was suppressed in SMCs cultured on floating collagen when compared to those cultured on attached
collagen gels. No significant differences between the total
number of TCA precipitable counts were noted between
cells cultured on attached (1.10 × 106 cpm/culture, SEM ± 9.6 × 104, P < 0.05) versus floating collagen (1.13 × 106
cpm/culture, SEM ± 1.59 × 104, P < 0.05), indicating that
despite suppression of TN-C protein synthesis, general
protein synthesis at this time point was unaffected.
Fig. 1.
Tenascin-C and matrix metalloproteinase-2 (MMP-2)
expression in vascular smooth muscle cells. (A) Representative
photomicrograph showing SMC morphology on attached type I
collagen gels and on collagen gels that have been released for 2 h
into the culture medium. (B) Autoradiograph (representative of
two different experiments) showing immunoprecipitated TN protein from [35S]methionine/cysteine-labeled SMC lysates harvested from attached and floating type I collagen gels in SFM
with 50 ng/ml EGF at 24 h. Synthesis of two TN-C protein isoforms of apparent molecular masses 220 and 180 kD, is suppressed in cells cultured on floating collagen. (C) Gelatin zymography was used to examine the levels and activity of gelatinases
present in conditioned medium (CM) harvested from SMCs cultured on attached and floating collagen. Fresh SFM with 50 ng/ml
EGF was added for 4 h before its collection at 4, 8, 12, and 24 h.
Equal volumes of concentrated CM were then analyzed by gelatin substrate zymography in nonreducing 10% polyacrylamide gels containing 0.1% gelatin. A zymogram (representative of
three different experiments) shows that on attached and floating
collagen, SMCs secrete gelatinases with apparent molecular
masses of 68 and 57 kD, and that on floating collagen, the activity
is lower. (D) Densitometry of the 68- and 57-kD gelatinases
shown in C. (E) Western immunoblot for the latent form of
MMP-2 in conditioned medium harvested at 24 h from SMCs cultured on attached and floating collagen shows that expression of
the 72-kD proform of this enzyme (equivalent to the 68-kD species under nonreducing conditions on the zymogram) is suppressed under floating conditions. Bar, 120 µm.
[View Larger Versions of these Images (98 + 37K GIF file)]
). Thus, our data indicate that the gelatinases detected by zymography may represent MMP-2. Their identity as MMPs was substantiated by the abolishment of gelatinase activity after incubation of zymograms with EDTA
or GM6001 (data not shown) and Western immunoblot
analysis, which showed that expression of the pro-form of
MMP-2 (which migrates at 72 kD under denaturing and reducing conditions) is suppressed on floating collagen
(Fig. 1 E).
3 Integrins Positively
Regulate Smooth Muscle Cell Tenascin-C Production
). At a functional level, this leads to exposure of cryptic Arg-Gly-Asp (RGD) sites within the native
type I collagen molecule that bind the
v
3 integrin receptor
to promote cell survival (Davis, 1992
; Montgomery et al.,
1994
). This integrin effect may also be achieved by culturing
cells on heat-denatured collagen (Montgomery et al., 1994
).
Since increased SMC TN-C expression correlates with increased MMP expression and activity, we hypothesized that
degradation of native type I collagen by MMPs may upregulate SMC TN-C expression through
3 integrin ligation.
; Strauss et al., 1996
). Immunoprecipitation of TN-C
from radiolabeled cell lysates showed that inhibition of
MMP activity results in suppression of TN-C protein synthesis (Fig. 2 A). In addition, the ability of this inhibitor to
suppress TN-C protein synthesis is not due to cytotoxicity
since there were no significant differences between the total number of TCA precipitable counts in radiolabeled
control (3.18 × 106 cpm/culture, SEM ± 2.12 × 105, P < 0.05) and GM6001-treated cultures (3.75 × 106 cpm/culture, SEM ± 1.63 × 105, P < 0.05) at the time point examined.
Fig. 2.
Regulation of tenascin-C protein expression by matrix
metalloproteinases and 3 integrins. (A) Immunoprecipitation of
TN-C protein from [35S]methionine/cysteine-labeled SMCs cultured on native type I collagen gels in serum-free control medium
with 50 ng/ml EGF and 0.4% DMSO, or in control medium supplemented with 1 or 2 µM of GM6001, an MMP inhibitor. Inhibition of MMP activity with GM6001 leads to a marked decrease in
TN-C protein synthesis. Positions of molecular mass standards in
kD are indicated on the left. (B) Western immunoblotting for
TN-C protein from SMCs cultured in SFM with 50 ng/ml EGF on native and heat-denatured type I collagen gels. A Western immunoblot shows that expression of two TN-C isoforms of apparent
molecular masses 220 and 180 kD is increased in cells cultured on
denatured collagen. (C) Western immunoblot of TN-C protein
from SMCs cultured on heat-denatured type I collagen gels in
control medium supplemented with either 0.4% DMSO or 2 µM
of GM6001. Inhibition of MMP activity with GM6001 had no effect on TN-C protein expression. (D) Effect of
3 integrin blockade
on TN-C protein expression on heat-denatured collagen. Immunoprecipitation of TN-C protein from [35S]methionine/cysteine-
labeled SMCs cultured on native type I collagen gels in the presence of control IgG or with anti-
3 antibody shows that blocking
3 integrins inhibits TN-C protein expression on native type I collagen. (E) Effect of
3 integrin blockade on TN-C protein expression on heat-denatured collagen. Western immunoblot analysis
of lysates derived from cells cultured on heat-denatured collagen
in the presence of control IgG, or with anti-
3 integrin antibody
shows that blocking
3 integrins inhibits TN-C protein expression
on denatured collagen. Arrows indicate the presence of the 220-kD
(upper band) and 180-kD (lower band) TN-C isoforms.
[View Larger Version of this Image (37K GIF file)]
3 integrins, we
used a function-blocking anti-
3 antibody. Western immunoblots showed decreased TN-C protein expression in the
presence of the anti-
3 antibody when compared with IgG-treated cultures on both native (Fig. 2 D) and proteolyzed
collagen. (Fig. 2 E).
), we next determined the functional
consequences of suppressing MMPs on EGF-dependent
SMC morphology, growth, and survival, in the presence
and absence of exogenously added TN-C. Inhibition of MMPs with GM6001 in SMCs cultured on attached collagen resulted in cellular rounding (data not shown) and a
significant decline in SMC number, whereas inclusion of
exogenous TN-C in the collagen gels prevented this decrease (Fig. 3 A) and restored their characteristic elongated morphology (data not shown). Similarly, on floating collagen, in which MMPs and TN-C expression are suppressed, SMC survival is diminished (Fig. 3 B) via apoptosis as determined by oligonucleosomal DNA fragmentation assays (Fig. 3 C). In contrast, SMC numbers were
unaffected (Fig. 3 B), and apoptosis was suppressed in
cells cultured on floating collagen gels supplemented with exogenous TN-C protein (Fig. 3 C).
Fig. 3.
Matrix metalloproteinases and tenascin-C act as vascular smooth muscle cell survival factors. (A) Smooth muscle cells
(2 × 104 cells per dish) plated on type I collagen were maintained
in serum-free control medium with 0.4% DMSO and 50 ng/ml
EGF, or in control medium supplemented with 1 or 2 µM of
GM6001 and EGF. GM6001 treatment resulted in a significant
decline in SMC numbers by 48 h, whereas addition of exogenous
human TN-C protein (15 µg/ml) to attached collagen substrates
inhibited this effect. (B) Effect of exogenous TN-C on SMC numbers on floating collagen gels. A significant decline in SMC numbers is apparent on floating compared to attached collagen gels
(P < 0.05), whereas addition of TN-C suppresses this effect. Values shown in C and D represent mean ±SEM derived from three
experiments. The asterisk denotes a P < 0.05 difference from cell
numbers recorded on collagen gels in EGF-containing medium.
(C) Effect of TN-C on SMC apoptosis on floating collagen gels.
Smooth muscle cells plated on attached type I collagen gels (2 × 104 cells per dish) were maintained in SFM with EGF (50 ng/ml)
for 48 h or were floated in the same medium, either with or without addition of exogenous human TN-C (15 mg/ml). Genomic
DNA was isolated from each culture and 10 µg per sample was
analyzed on 1% agarose gels. DNA fragments comprised of
~180-bp multimers, which are indicative of apoptosis, were apparent on floating collagen gels. In contrast, no evidence of DNA
fragmentation was observed on either attached or TN-C-supplemented floating collagen. Positions of a standardized DNA ladder in kb are indicated on the right.
[View Larger Version of this Image (31K GIF file)]
v
3 Integrin
to Promote Epidermal Growth Factor-dependent
Smooth Muscle Cell Proliferation
v
3 integrin
(Prieto et al., 1993
; Sriramarao et al., 1993
). To determine
whether this integrin mediates TN-C/EGF-dependent
SMC growth, we evaluated the effect of a functional-blocking anti-
v
3 integrin monoclonal antibody (LM609)
on cell morphology and proliferation of SMC cultured on
collagen and TN-C-enriched substrates in the presence of
EGF. While LM609 had no effect on the stellate SMC
morphology observed on collagen, the more elongated
SMC morphology induced by TN-C was attenuated by this
antibody. Instead, SMCs failed to spread and remained
rounded (Fig. 4 A). LM609 had no significant effect on SMC
attachment to TN-C-enriched substrates (Fig. 4 B) or on cell number after culture of SMC on collagen in the presence of EGF (Fig. 4 C). In contrast, TN-C-dependent growth
in response to EGF was inhibited by LM609 (Fig. 4 C).
Fig. 4.
Effect of blocking
v
3 integrins on tenascin-
C-dependent smooth muscle cell morphology, attachment efficiency, and survival. (A) Representative phase
contrast photomicrographs
of SMCs plated in SFM on
collagen and TN-C-supplemented collagen gels with
control IgG (15 µg/ml) or
with an anti-
v
3 integrin antisera (LM609; 15 µg/ml).
IgG and LM609 treatment
had no effect on the stellate morphology produced by the
collagen substrate. In contrast,
the more elongated SMC morphology observed on control-treated TN-C-enriched substrates was abrogated by
inclusion of LM609, which
prevented cells from spreading, resulting in a rounded
morphology. (B) Effect of
LM609 antisera on SMC attachment to TN-C-supplemented type I collagen gels.
By 6 h after plating, no significant differences in attachment efficiency were noted
between cells plated in SFM on TN-C-supplemented collagen gels in either the presence of control IgG or
LM609 antisera. (C) Effect
of blocking SMC
v
3 integrins with LM609 antisera on
EGF-dependent SMC cell
growth on TN-C-supplemented collagen gels. Control and LM609 treatment
produced no differences in
SMC number after culture
for 48 h on collagen gels in SFM with 50 ng/ml EGF. The significant (P < 0.05) increase in SMC growth observed on TN-supplemented
collagen gels in the presence of EGF was attenuated by LM609 antisera. Values represent mean ±SEM from three different experiments. (The asterisk denotes P < 0.05 difference from IgG control level on type I collagen.) Bar, 150 µm.
[View Larger Versions of these Images (118 + 13K GIF file)]
v
3 integrin interactions on SMC
shape and EGF-dependent proliferation provided us with
the rationale for investigating the organization of the filamentous actin cytoskeleton and the distribution of EGF-Rs and tyrosine-phosphorylated proteins. Accordingly, we
used immunofluorescence microscopy to evaluate the distribution of F-actin in SMCs cultured on collagen gels supplemented with exogenous TN-C, and we determined
whether this pattern was further altered by EGF (Fig. 5
A). Rhodamine-phalloidin staining revealed a longitudinal
distribution pattern of F-actin stress fibers in SMCs cultured on collagen, which became more cortical after addition
of EGF. On TN-C-supplemented collagen gels, however,
high-intensity F-actin staining was observed at the cell periphery, and this pattern intensified after addition of EGF.
Fig. 5.
Tenascin-C modifies
the patterns of distribution for
filamentous actin, epidermal
growth factor receptors, tyrosine-phosphorylated proteins,
and vinculin. (A) Representative
immunofluorescence photomicrographs (from two different
experiments) showing distribution patterns for F-actin, EGF-Rs,
and tyrosine-phosphorylated
(P-Tyr) proteins in SMC cultured in SFM (+/- 50 ng/ml EGF
for 30 min) on collagen alone or
on TN-C (15 µg/ml) supplemented
collagen substrates. Rhodamine-phalloidin staining of SMC on
collagen revealed a longitudinal
F-actin stress fiber pattern of
distribution that was more cortical after treatment with EGF.
Immunofluorescent staining for
EGF-Rs and tyrosine-phosphorylated proteins was diffuse in
SMC cultured on collagen alone
and increased modestly after addition of EGF. In contrast, SMC
cultured on TN-C-supplemented collagen gels showed high-intensity F-actin staining in regions that often overlapped with clusters of EGF-Rs and tyrosine-phosphorylated proteins. After
addition of EGF to TN-C-treated
cultures, the levels and distribution of EGF-Rs remained pronounced, and high levels of tyrosine-phosphorylated proteins
were evident throughout the
cell. (B) Representative double
immunofluorescence photomicrographs showing codistribution of vinculin, F-actin, EGF-Rs,
and tyrosine-phosphorylated (P-Tyr) proteins in SMC cultured in SFM on collagen supplemented with exogenous human TN-C protein. Bars: (A) 20 µm; (B) 5 µm.
[View Larger Versions of these Images (35 + 59K GIF file)]
), we then assessed whether TN-C-dependent alterations in the organization of the F-actin
cytoskeleton may be accompanied by EGF-R clustering, a
prerequisite for EGF-dependent signaling, including ligand-dependent tyrosine phosphorylation (Heldin, 1995
). Immunofluorescent studies revealed a diffuse pattern of
EGF-R staining and tyrosine phosphorylation in SMCs
cultured on collagen alone (Fig. 5 A). By contrast, in
SMCs cultured on TN-C-enriched collagen gels, prominent EGF-R clustering was observed, especially at the cell
periphery. After addition of EGF to collagen gels, a modest increase in both EGF-R clustering and tyrosine phosphorylation was apparent, whereas in TN-C-treated cultures, EGF-R clustering remained pronounced and high
levels of tyrosine phosphorylation were evident within the
nucleus and at a relatively lower level at the cell periphery.
Furthermore, double immunostaining experiments with an
antivinculin antibody established that TN-C-treated SMCs
contained more focal adhesion complexes, which were
larger than those produced on type I collagen alone (data
not shown). Most strikingly, exogenous TN-C promoted
accumulation of F-actin, EGF-Rs, and tyrosine-phosphorylated proteins within these focal adhesion sites (Fig. 5 B).
Fig. 6.
Tenascin-C potentiates ligand-dependent epidermal
growth factor-receptor tyrosine phosphorylation. (A) Effect of
TN-C on EGF-R protein synthesis. Representative autoradiograph (from two different experiments) showing immunoprecipitation of EGF-R protein from [35S]methionine-labeled SMCs cultured on native type I collagen gels (+/ 15 µg/ml TN-C) in SFM
indicates that TN-C has no effect on the levels of EGF-R protein
synthesis compared to SMCs cultured on collagen alone. (B) Effect of TN-C on tyrosine phosphorylation in SMCs cultured on
collagen and TN-C-supplemented gels in SFM, or after addition
of EGF (50 ng/ml) for 10 and 30 min. A Western immunoblot
(representative of two different studies) was performed on membrane-enriched fractions (15 µg per sample) using an antibody against tyrosine-phosphorylated proteins. Note that relative to SMCs cultured on collagen alone, contact with exogenous TN-C
led to an increase in basal levels of tyrosine phosphorylation. In response to EGF, qualitative and quantitative increases in tyrosine-phosphorylated proteins were observed on both collagen
and TN-C, with changes being more pronounced on TN-C-enriched
gels, including species at 170 and 125 kD. Positions of molecular
mass standards in kD are indicated on the right. The 170-kD species
may represent the EGF receptor, and the 125-kD species the focal adhesion kinase. (C) The 170-kD tyrosine-phosphorylated
protein shown in B represents the EGF-R. Epidermal growth factor receptors were immunoprecipitated from 15 µg of membrane-enriched SMC fractions in cells cultured as in B. Epidermal
growth factor immunoprecipitates were analyzed for evidence of
tyrosine phosphorylation using Western immunoblotting with an
antiphosphotyrosine antibody. After addition of EGF at 10 min,
a transient increase in the levels of tyrosine-phosphorylated EGF-Rs occurs on collagen and TN-C-supplemented gels, with
greater levels observed on TN-C-supplemented collagen substrates.
[View Larger Version of this Image (40K GIF file)]
3 Integrins Regulate Tenascin-C-dependent
Epidermal Growth Factor Receptor Clustering and
Ligand-dependent Tyrosine Phosphorylation
3 integrin SMC interactions on EGF-R clustering and tyrosine
phosphorylation. Smooth muscle cells were preincubated
with an anti-
3 integrin antibody before plating on TN-
C-supplemented collagen gels. As with the
v
3 functional-blocking antibody experiments, the anti-
3 integrin
antibody prevented cell spreading on TN-C (data not
shown). We therefore used the anti-
3 integrin antibody
in subsequent studies (although similar results would be
expected with the
v
3 antibody, LM609) and showed inhibition of TN-C-dependent EGF-R clustering (Fig. 7 A).
Immunoprecipitation of SMC membrane-enriched fractions followed by Western immunoblotting with an antiphosphotyrosine antibody showed that upon addition of
EGF, EGF-R activation on TN-C-enriched collagen substrates was abrogated in cells pretreated with the function-blocking anti-
3 integrin antibody (Fig. 7 B).
Fig. 7.
The role of 3 integrins in tenascin-C-dependent epidermal growth factor receptor clustering and ligand-dependent
receptor activation. (A) Representative immunofluorescence
photomicrographs for EGF-R in SMCs cultured on TN-C (15 µg/
ml) supplemented collagen gels in the presence of control IgG or
with an anti-
3 integrin antibody that prevented the formation of
EGF-R clusters. (B) Effect of blocking
3 integrins on ligand-dependent EGF-R tyrosine phosphorylation in SMCs cultured on
collagen gels supplemented with TN-C. Western immunoblot
(representative of two different studies) of membrane preparations from SMCs treated with control IgG, with anti-
3 integrin
antisera on TN-C-supplemented collagen in SFM alone, or in
SFM with EGF (50 ng/ml) for 10 and 30 min. Epidermal growth factor receptors were immunoprecipitated from 15 µg of membrane-enriched SMC fractions and were analyzed for evidence of
tyrosine phosphorylation using Western immunoblotting. Note
that the anti-
3 integrin antibody prevents the transient increase
in EGF-R tyrosine phosphorylation observed in TN-treated cultures after addition of EGF at 10 min. Bar, 17 µm.
[View Larger Versions of these Images (17 + 32K GIF file)]
3 Integrin Cross-Linking Promotes Clustering and
Activation of Epidermal Growth Factor Receptors
3 integrins on SMC
surfaces, in the absence of exogenous TN-C, promotes
EGF-R clustering and ligand-dependent phosphorylation
of EGF-Rs, anti-
3 integrin antibody cross-linking studies
were performed. Smooth muscle cells cultured on type I
collagen gels were treated with either a combination of
anti-
3 integrin and secondary goat anti-mouse IgG F(ab
)2 antibodies or with primary mouse monoclonal anti-
3
integrin antibody alone. Immunofluorescent microscopy
revealed extensive
3 integrin clusters on SMC surfaces
treated with primary and secondary antibodies, whereas a
more diffuse pattern of
3 integrins was apparent in cultures treated with primary antibody alone (Fig. 8 A). Immunofluorescent microscopy with an anti-EGF-R antibody
demonstrated that cross-linking the
3 integrin receptor
leads to clustering of EGF-Rs, which appeared diffuse in
SMCs treated with primary antibody alone (Fig. 8 A).
Fig. 8.
Effect of cross-linking 3 integrins on epidermal growth factor receptor clustering. (A) Representative immunofluorescence micrographs for
3 integrins and EGF-Rs. Smooth muscle cells cultured on collagen substrates in SFM were preincubated for 60 min
with an anti-
3 integrin antibody (150 µg/ml) and then for 60 min with SFM alone or with an anti-F(ab
)2 IgG (20 µg/ml) to promote
cross-linking. Immunodetection of
3 integrins and EGF-Rs indicates that cross-linking
3 integrins promotes EGF-R clustering. (B) Effect
of cross-linking
3 integrins on ligand-dependent tyrosine phosphorylation of EGF-Rs. Tyrosine phosphorylation of EGF-Rs in response to EGF was examined in SMC cultures treated with anti-
3 integrin antibody alone or in cultures incubated sequentially with an
anti-
3 integrin antibody and anti-F(ab
)2 IgG. Epidermal growth factor receptor immunoprecipitates were analyzed by Western immunoblotting using an antiphosphotyrosine antibody, which indicated that cross-linking
3 integrins potentiates ligand-dependent EGF-R
activation. (C) Densitometry of activated epidermal growth factor Western immunoblots (as shown in B) shows that a significant (*P < 0.05) increase in ligand-dependent tyrosine phosphorylation occurs after
3 integrin cross-linking. Values represent mean ±SEM from
three different experiments. Bar, 25 µm.
[View Larger Versions of these Images (17 + 16 + 14K GIF file)]
3 integrin-dependent
EGF-R clustering on tyrosine phosphorylation of EGF-Rs, EGF was added to SMCs, which had been pretreated
either with a combination of mouse anti-
3 integrin and
secondary goat anti-mouse IgG F(ab
)2 antibodies, or with
anti-
3 integrin antibody alone. Immunoprecipitation of
EGF-Rs from SMC membrane fractions followed by Western immunoblotting with an antiphosphotyrosine antibody
showed that a significant increase in EGF-dependent activation of EGF-Rs resulted from cross-linking
3 integrin
receptors (Fig. 8, B and C).
DISCUSSION
; Hahn
et al., 1995
; Chen et al., 1996
; Jones and Rabinovitch, 1996
;
Jones et al., 1997
). However, remarkably little was known
about the nature of the factors that determine its expression in these tissues or the mechanism by which TN-C supports SMC growth. In this study, we demonstrate that
MMPs positively regulate TN-C protein expression and
that the mechanism likely involves exposure of cryptic
3
integrin-binding sites in type I collagen. We also show that
TN-C rescues SMCs from apoptosis and promotes EGF-dependent SMC survival through interactions with the
v
3 integrin receptor, which modulates cell shape and
EGF-dependent tyrosine phosphorylation. These studies therefore provide new evidence to show how remodeling
of the extracellular microenvironment by ECM-degrading
proteinases may influence integrin growth factor receptor
signaling functions and thus cell behavior. Fig. 9 shows a
hypothetical model for the regulation and function of TN-C
in SMCs based on our findings.
Fig. 9.
Hypothetical model for the
regulation and function of tenascin-C
in vascular smooth muscle cells. (A)
Vascular smooth muscle cells attach
and spread on native type I collagen
using 1 integrins. Under serum-free
conditions, the cells withdraw from the
cell cycle and become quiescent. (B)
Degradation of native type I collagen by matrix metalloproteinases (MMPs)
leads to the exposure of cryptic RGD
sites that preferentially bind
3 subunit-containing integrins. In turn, occupancy and activation of
3 integrins
signals the production of TN-C. (C) Incorporation of multivalent TN-C protein into the underlying substrate leads
to further aggregation and activation
of
3-containing integrins (
v
3), and to
the accumulation of tyrosine-phosphorylated (Tyr-P) signaling molecules and
actin into a focal adhesion complex
(FAC). Note that even in the absence of the EGF ligand, the TN-C-dependent reorganization of the cytoskeleton leads to clustering of actin-associated EGF-Rs. (D) Addition of EGF
ligand to clustered EGF-Rs results in
rapid and substantial tyrosine phosphorylation of the EGF-R and activation of downstream pathways culminating in the generation of nuclear
signals leading to cell proliferation.
[View Larger Version of this Image (41K GIF file)]
demonstrated that chick embryo fibroblasts cultured on attached collagen gels express TN-C protein, whereas in
floating cultures, TN-C synthesis is preferentially suppressed. This regulation occurs at the transcriptional level
and was mapped to a putative "ECM response element" in
the TN-C gene promoter that is distinct from the region
important for induction by serum. However, the reason for this differential TN-C production on attached and
floating collagen and the functional consequences of suppressing TN-C expression were not explored.
; Boudreau et al., 1995b
), and that these functions may depend
upon proteolysis of ECM and cell surface molecules (Fujii
and Imamaura, 1995; Brooks et al., 1996
). Since MMPs
and TN-C are often expressed at the same site within normal (Sympson et al., 1994
; Jones et al., 1995
) and injured remodeling tissues (Latijnhouwers et al., 1996
; Vaalamo et
al., 1996
), including blood vessels (Bendeck et al., 1994
;
Strauss et al., 1996
; Jones, P.L., J. Crack, and M. Rabinovitch. 1996. Mol. Biol. Cell. 7:418a), we examined the possibility that MMPs regulate SMC growth and viability
through the induction of TN-C. Accordingly, we established a role for MMPs in regulating TN-C expression and
SMC survival by showing that reducing MMP activity and
expression on floating collagen gels, or with a specific
MMP inhibitor, limits TN-C protein synthesis and SMC
growth and survival.
). For example,
MMP-2 exposes cryptic RGD ligands in native collagen,
which may bind
v
3 integrins to promote cell survival
(Montgomery et al., 1994
), an effect which can be mimicked by culturing cells on heat-denatured collagen (Davis,
1992
; Montgomery et al., 1994
). In the present study, we
demonstrate that on native type I collagen, MMPs promote TN-C protein expression via
3 integrins, and using
denatured collagen, we show that this substrate supports TN-C expression in a manner that still requires
3 integrins but is independent of MMPs. Thus, on attached type I
collagen gels, activated MMPs may generate two RGD-containing ligands, i.e., denatured collagen and TN-C, and in
this way, serve a dual function in cell survival. In addition,
recent studies indicate that MMP-2 directly interacts with
the
v
3 integrin receptor to form a functional complex
(Brooks et al., 1996
). Since the appearance of TN-C expression within remodeling tissues is restricted in a precise
spatial manner (Chiquet-Ehrismann et al., 1995
), it is also
tempting to speculate that induction of TN-C by MMPs
serves to localize this ECM component with its cognate
adhesion receptors, which include the
v
3 integrin (Prieto
et al., 1993
; Sriramarao et al., 1993
).
; Mooney et al., 1995
;
Ingber, 1993
; Roskelley et al., 1994
; Boudreau et al., 1996
;
Chen, 1997; Weaver et al., 1997
). Our results show that
suppression of endogenous TN-C occurs within a rounded
SMC population, in which EGF-dependent growth and
survival are limited, whereas addition of exogenous TN-C
to these cultures is associated with the restoration of an elongated cell shape, the reestablishment of an EGF-dependent
growth response, and the prevention of apoptosis. Also,
using functional-blocking anti-
3 integrin antibodies, we
show that EGF-R signaling is attenuated in SMCs that fail
to spread on TN-C-enriched collagen substrates. These
data strongly indicate that TN-C-dependent cell shape alterations may impact cell growth and survival functions in
SMCs. Consistent with this idea, suppression of TN-C expression in mammary epithelial cells is associated with cellular rounding (Jones et al., 1995
) and repression of
growth-related genes including c-myc (Boudreau et al.,
1996
). Furthermore, we have shown that SMCs plated on nonadhesive substrates remain rounded and are unable to
proliferate in response to EGF, whereas on tissue culture
plastic, SMC spread and are able to grow (Jones, P.L., and
M. Rabinovitch, unpublished observations).
), whereas cells in mechanically relaxed matrices become arrested in G0 and are less responsive to addition of serum or purified growth factors (Nishiyama et al., 1991
). Mechanistically, autophosphorylation
of PDGF and EGF-Rs in fibroblasts is decreased in mechanically relaxed collagen (Lin and Grinnell, 1993
),
whereas
1 integrin-dependent autophosphorylation of
platelet derived growth factor (PDGF
) receptors is induced in fibroblasts in response to the application of external tension (Sundberg and Rubin, 1996
). It is therefore
possible that mechanical induction of TN-C and/or other
ECM components might be important in the activation of
a more extensive repertoire of growth factor receptors. Indeed, we have previously shown that TN-C cooperates
with another soluble growth factor, bFGF, to facilitate
SMC proliferation (Jones and Rabinovitch, 1996
). Also,
the cooperative effect between TN-C, EGF, and bFGF
does not appear to be restricted to a single cell type, or cell
surface receptor. Chung et al. (1996)
showed that cell surface annexin II, which also acts as a TN-C receptor (Chung and Erickson, 1995), mediates TN-C/bFGF-dependent
endothelial cell growth, whereas End et al. (1992)
showed
that TN-C and EGF stimulate proliferation of Swiss fibroblasts, NIH-3T3 cells and SMCs isolated from spontaneously hypertensive rats. Our present studies therefore confirm the idea that TN-C cooperates with soluble growth
factors to promote cell proliferation and extend these studies by providing a mechanism for these cooperative effects that is based on ECM-dependent cell shape changes
and cross-talk between integrins and growth factor signaling pathways.
). In turn, EGF-R
activation catalyzes the tyrosine phosphorylation of several
protein substrates, including the receptor itself (Hunter
and Cooper, 1988). In the present study, we show for the
first time that the
v
3 integrin mediates TN-C/EGF-dependent growth, and that this interaction leads to cell shape
changes that are underscored by a redistribution of filamentous actin, clustering of EGF-Rs, and accumulation of tyrosine-phosphorylated proteins at the cell periphery
within focal adhesion contacts, as well as increased activation of EGF-Rs after addition of EGF. Moreover, these
TN-C-dependent effects on EGF-R clustering and tyrosine phosphorylation could be mimicked by cross-linking SMC
3 integrins. It is therefore possible that other
ECM components that bind
3 integrins, e.g., vitronectin and proteolysed collagen, which have also been implicated
in vascular pathobiology (Montgomery et al., 1994
; Brooks
et al., 1996
), could function in a manner similar to TN-C.
Indeed, a recent report shows that denatured monomeric
collagen stimulates increased focal adhesion contact
formation and PDGF-BB-dependent SMC proliferation (Koyama et al., 1996
), whereas polymerized collagen inhibits growth by upregulating cyclin-dependent kinase inhibitors including p27kip1 and p21Cip1/Waf1.
; Burridge and Chrzanowska-Woodnicka, 1996
). In addition, a number of studies have provided evidence in favor of a direct role for the
actin cytoskeleton in EGF-R signaling. For example, the
EGF-R has been shown to be an actin-binding protein
(den Hartigh et al., 1992
), and from a functional standpoint, this association leads to the accumulation and activation of EGF-R substrates at the binding site (Gronowski
and Bertics, 1993; Diakonova et al., 1995
). However, the
functional consequences of these types of interactions and
the identity of cell surface receptors involved had not been
explored. Our results strongly indicate that ligation of substrate-bound TN-C to SMC
v
3 integrins leads to the formation of specialized cytoskeletal structures enriched with
tyrosine-phosphorylated proteins that together favor the recruitment and clustering of actin-associated high-affinity
EGF-Rs. Addition of EGF to these cells, which are now
both physically and biochemically primed, would allow the
generation of an optimal and ECM-specific mitogenic response.
; Jones et al., 1997
), we suggest that TN-C and its receptors may be prime therapeutic targets for inhibiting
SMC growth that is associated with vascular disease.
Received for publication 18 December 1996 and in revised form 2 July 1997.
Address all correspondence to Marlene Rabinovitch, M.D., Division of Cardiovascular Research, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, Canada MSG 1X8. Tel: (416) 813-5918. Fax: (416) 813-7480. e-mail: mr{at}sickkids.on.caWe would like to thank Drs. D. Cheresh, I. Lax, M. Silverman and M. Ailenburg, and Glycomed Inc. (GM6001 inhibitor) for providing reagents. We are also indebted to Claire Coulber, Joan Jowlabar, and Susy Taylor for their help in preparing this manuscript.
This study was supported by program grant T2229 from the Heart and Stroke Foundation of Canada.
ECM, extracellular matrix; EGF-R, epidermal growth factor receptor; MMP, matrix metalloproteinase; SFM, serum-free medium; SMC, smooth muscle cell; TN-C, tenascin-C.
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