* DIBIT, Department of Biological and Technological Research, San Raffaele Scientific Institute, 20132 Milano, Italy; Department of Biomedical Sciences and Human Oncology, University of Torino School of Medicine, 10126 Torino, Italy; and § Department of Pathology, Clinical and Experimental Medicine, University of Udine School of Medicine, 33100 Udine, Italy
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Abstract |
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Integrin activation is a multifaceted phenomenon leading to increased affinity and avidity for matrix
ligands. To investigate whether cytokines produced
during stromal infiltration of carcinoma cells activate
nonfunctional epithelial integrins, a cellular system of
human thyroid clones derived from normal glands
(HTU-5) and papillary carcinomas (HTU-34) was employed. In HTU-5 cells, v
3 integrin was diffused all
over the membrane, disconnected from the cytoskeleton, and unable to mediate adhesion. Conversely, in
HTU-34 cells,
v
3 was clustered at focal contacts
(FCs) and mediated firm attachment and spreading.
v
3 recruitment at FCs and ligand-binding activity, essentially identical to those of HTU-34, occurred in
HTU-5 cells upon treatment with hepatocyte growth
factor/scatter factor (HGF/SF). The HTU-34 clone secreted HGF/SF and its receptor was constitutively tyrosine phosphorylated suggesting an autocrine loop responsible for
v
3 activated state. Antibody-mediated
inhibition of HGF/SF function in HTU-34 cells disrupted
v
3 enrichment at FCs and impaired adhesion.
Accordingly, activation of
v
3 in normal cells was
produced by HTU-34 conditioned medium on the basis
of its content of HGF/SF. These results provide the first
example of a growth factor-driven integrin activation mechanism in normal epithelial cells and uncover the
importance of cytokine-based autocrine loops for the
physiological control of integrin activation.
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Introduction |
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ADHESION to neighboring cells and the extracellular
matrix (ECM)1 plays a crucial role in different biological phenomena, including cell motility and tumor invasion (Juliano and Varner, 1993; Chapman, 1997
),
differentiation (Adams and Watt, 1993
; Lin and Bissell, 1993
; Gumbiner, 1996
), and survival (Frisch and Francis,
1994
). The malignant behavior of carcinoma cells is not
simply characterized by alteration or loss of growth control, a feature shared with benign neoplasms, but also by
the ability to weaken tissue constraints and invade foreign
districts, where cancer cells may migrate, proliferate, and
survive. This xenophilic tendency is fostered by cooperation among ECM molecules, proteases, growth factors (GFs), and the adhesion receptors expressed on the surface of the invading cells, which together provide signals
controlling the organization of the cytoskeleton (Clark
and Brugge, 1995
; Yamada and Miyamoto, 1995
; Brooks
et al., 1996
; Wei et al., 1996
).
Physiological interactions between normal epithelial
cells and the underlying basal lamina, as well as recognition of matrix components by carcinoma cells during stromal infiltration are mediated by the integrin family of adhesion receptors, a class of transmembrane noncovalently
associated glycoprotein heterodimers composed of one and one
chain (Hynes, 1992
; Sonnenberg, 1993
). Conceivably, migration of epithelial neoplastic cells within
stromal tissues involves changes in the expression, topography, cytoskeletal association, and signaling properties of
the integrin repertoire. In fact, many in vivo and in vitro
studies have reported surface modifications of integrin
levels, or even neo-expression of some integrins, in carcinoma versus normal cells (Plantefaber and Hynes, 1989
;
Zutter et al., 1995
; Serini et al., 1996
; for a comprehensive review see Ben-Ze'ev, 1997
). Such modifications might be
driven either by the ECM itself (Langhofer et al., 1993
;
Rabinovitz and Mercurio, 1996
), by GFs secreted by stromal cells and stimulating the invasive neoplastic elements
in a paracrine fashion (Klemke et al., 1994
; Doerr and
Jones, 1996
), or even by cytokines synthesized de novo by
carcinoma cells and acting back on the tumor mass via an
autocrine circuit (Aasland et al., 1988
; Bachrach et al.,
1988
; Mizukami et al., 1991
).
Cellular attachment to immobilized ECM ligands commonly results in cytoskeletal reorganization and clustering
of integrins at discrete adhesive sites known as focal contacts (FCs). In these specialized structures, an array of
submembranous proteins ranging from structural molecules to regulatory enzymes forms a multimolecular complex linking the actin microfilament network with integrins and, hence, with the ECM (Burridge and Chrzanowska-Wodnicka, 1996). The formation of FCs triggers signal cascades that act in concert with GF-activated transduction
pathways and can alter gene expression (Clark and
Brugge, 1995
). Under given conditions, intracellular signals that originate at FCs or at downstream targets result in modulation of the affinity and/or avidity of certain integrins for extracellular ligands, a process termed activation
or inside-out signaling (Hynes, 1992
).
Our goal was to investigate how variations in integrin
composition and GF receptor activation correlate with
changes in cell adhesion in carcinoma versus normal elements. We assumed that the tuning of the integrin adhesive machinery occurring when tumor cells invade surrounding tissues might include not only changes in
quantity but also changes in quality such as conversion of integrins from a dormant, nonadhesive state into an active
one endowed with high adhesive capabilities. Specifically,
we postulated that such conversion could be induced by
one or more GFs acting within the neoplastic environment. The cellular model used in our experiments was a
panel of human thyroid clonal strains corresponding to
normal and malignant in vivo cell populations. These
clones can form epithelial colonies and, when cultured in
three-dimensional gels, develop aggregates whose biological functions, differentiation parameters, and morphological architecture are strictly related to the in vivo counterpart (Curcio et al., 1994; Perrella et al., 1997
; De Filippi, R.,
P.C. Marchisio, G. Serini, and L. Trusolino, manuscript in
preparation). Such a feature allows exhaustive comparisons between normal and carcinoma cells and is usually
limited or compromised in established cell lines, where relationship studies of normal versus transformed phenotypes appear to be extremely difficult.
In this report, we provide evidence that the v
3 integrin is expressed by normal cells in a latent state characterized by its inability to form cytoskeletal connections and to
promote cell adhesion to ECM ligands. In contrast,
v
3
is highly enriched at FCs of carcinoma cells and mediates
tight adhesion. We also demonstrate that the multifunctional cytokine hepatocyte growth factor/scatter factor
(HGF/SF), but not other serum GFs, is an autocrine factor
for carcinoma cells and show that the signaling pathway
stimulated by HGF/SF, when elicited in normal cells, can fully recapitulate the adhesive pattern of neoplastic elements. Indeed, no connections have been ever studied between a specific GF and activation of the adhesive capabilities of an integrin in normal epithelial cells under
physiological conditions. Here we unravel such a functional interplay and underscore the importance of autocrine production of GFs for the integrin-dependent invasive behavior of carcinoma cells and, possibly, for the
activation state of integrins in general.
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Materials and Methods |
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Cell Cultures
Clonal strains from human normal thyroid (HTU-5) and papillary carcinoma (HTU-34) were obtained and cultured as previously described
(Curcio et al., 1994; Perrella et al., 1997
). In brief, tissue samples from
pathological specimens of different patients were freed from adherent
connective tissue, cut into small pieces, washed in Ca2+- and Mg2+-free
HBSS, and enzymatically digested with a solution consisting of 20 U/ml
collagenase (CLSPA; Worthington Biochemical Corp., Freehold, NJ),
0.75 mg/ml trypsin (1:300; GIBCO BRL, Gaithersburg, MD), and 2%
heat-inactivated dialyzed chicken serum (GIBCO BRL), in Ca2+- and
Mg2+-free HBSS. Cell suspensions were collected after a 2 h digestion and
seeded onto 100-mm plastic tissue culture dishes (Falcon; Becton Dickinson, Lincoln Park, NJ). Culture medium was a modified F-12 further varied to contain 0.48 mM MgCl2, 3 mM KCl, 5% Fetal Calf Serum (GIBCO
BRL), 1 mg/ml Na-insulin (Elanco, Indianapolis, IN), 5 µg/ml bovine
transferrin (GIBCO BRL), 0.01 mM hydrocortisone, 2 ng/ml selenous
acid, 3 pg/ml triiodothyronine (all from Sigma Chemical Co., St. Louis,
MO), 75 µg/ml bovine hypothalamus extracts and 5 µg/ml bovine pituitary extracts (Pel Freez Biologicals, Rogers, AR). The purity of both
strains was assessed by examining the expression of thyroid-specific molecular markers (thyroglobulin, thyroperoxidase, TTF-1, and PAX-8) and
by evaluating thyrotropin-dependent c-AMP production and thymidine
incorporation. This in vitro profile was found to correlate with the degree
of differentiation of the starting specimen and with the pathological diagnosis (Perrella et al., 1997
). Throughout the experiments, only cells from
the 2nd to the 5th passage were used.
Antibodies
The integrin-specific mAbs used in this study (with the investigators who
provided them) were as follows: MAR4 against 1 and MAR6 against
6
(from Sylvie Ménard, Istituto Nazionale Tumori, Milano, Italy; Bottini et
al., 1993
); F2 against
3 (from Luciano Zardi, Istituto Scientifico per lo
Studio e la Cura dei Tumori, Genova, Italy); L230 against
v (from Paola
Defilippi, Dipartimento di Genetica, Biologia e Chimica Medica, University of Torino, Italy); AA3 against
4 (from Vito Quaranta, Scripps Research Institute, La Jolla, CA); VIPL-2 against
3 (from Walter Knapp,
Institüt für Immunologie der Universität, Vienna, Austria); IA9 against
5 (from Martin Hemler and Renata Pasqualini, Dana-Farber Cancer Institute, Boston, MA). Other mAbs against integrin subunits were commercially obtained: Gi9 against
2 and SAM-1 against
5 (Immunotech, Marseille, France); a rabbit polyclonal antiserum against
3 and the function-blocking mAb LM609 against the integrin complex
v
3 (Chemicon
International Inc., Temecula, CA). The inhibitory mAb AIIB2 against
1
was provided by Caroline H. Damsky (Department of Stomatology, University of California at San Francisco, CA). Rabbit polyclonal antisera
against
1 and
v were, respectively, from Ivan de Curtis (DIBIT, Istituto
Scientifico San Raffaele, Milano, Italy) and Guido Tarone (Dipartimento
di Genetica, Biologia e Chimica Medica, University of Torino, Italy).
mAb VIN11.5 against vinculin was from Sigma Chemical Co. The C-28
rabbit antiserum against human HGF/SF receptor, used in immunoprecipitation experiments, was purchased from Santa Cruz Biotechnology
(Santa Cruz, CA); the mAb DQ-13 against human HGF/SF receptor, used
in Western blotting analyses, and the 4G10 anti-phosphotyrosine mAb
were from Upstate Biotechnology Inc. (Lake Placid, NY). 1W53, a neutralizing sheep antiserum directed against human HGF/SF, was produced
in the laboratory of Ermanno Gherardi (Imperial Cancer Research Fund,
Cambridge University Medical School, UK) and kindly supplied by Paolo
Amati and Sergio Anastasi (Dipartimento di Biotecnologie Cellulari ed Ematologia, Università "La Sapienza", Rome, Italy). The neutralizing activity was titrated in scatter assays on MDCK cells after HGF/SF stimulation and found to be optimal at a 1:80 dilution.
Cytokines
HGF/SF and TGF1 were purchased respectively from R & D Systems
Inc. (Minneapolis, MN; Van der Voort et al., 1997
), and Boehringer Mannheim GmbH (Mannheim, Germany). EGF was kindly donated by Laura Beguinot (DIBIT, Milano, Italy). Insulin and insulin-like growth factor
were a generous gift of Franco Folli (Divisione Universitaria di Medicina
Interna, Istituto Scientifico San Raffaele, Milano, Italy).
Immunoprecipitation and Western Blotting
Immunoprecipitations were carried out on surface-biotinylated cells as
previously described (Rabino et al., 1994). In brief, confluent monolayers
were washed three times at 4°C with Hank's balanced salt biotinylation
buffer (HBB), pH 7.4, consisting of 1.3 mM CaCl2, 0.4 mM MgSO4, 5 mM
KCl, 138 mM NaCl, 5.6 mM D-glucose, and 25 mM Hepes, pH 7.4. Sulfosuccinimido biotin (Pierce Chemical Co., Rockford, IL) was made 0.5 mg/
ml in HBB and applied to the cells for 20 min at 4°C. The biotin solution
was then removed and replaced with fresh biotin solution for another 20 min. The reaction was stopped by incubating four times at 4°C with Minimal Essential Medium containing Hank's balanced salts, 0.6% BSA, 20 mM Hepes, pH 7.4. After three washes in cold HBB, cells were lysed for
30 min at 4°C in a buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl,
0.1% SDS, 1% Triton X-100, 0.5% sodium-deoxycholate, 5 mM EGTA,
50 mM NaF, and 5 mM MgCl2, supplemented with various phosphatase
and protease inhibitors (leupeptin, pepstatin, aprotinin, PMSF, soybean
trypsin inhibitor, and sodium-orthovanadate). Extracts were centrifuged
at 15,000 rpm for 30 min at 4°C, and supernatant protein content was normalized with the BCA Protein Assay Reagent Kit (Pierce Chemical Co.).
Cell lysates were precleared onto protein A-Sepharose CL-4B (Pharmacia Biotech Sverige, Uppsala, Sweden) and rotated 2 h at 4°C with different mAbs. Immunocomplexes were collected with affinity-purified rabbit
anti-mouse IgG (Pierce Chemical Co.) coupled to protein A-Sepharose.
After several washes with lysis buffer, the final pellets were eluted in boiling Laemmli buffer and proteins were electrophoresed on 8% SDS-PAGE. Samples were transferred onto Immobilon-PTM filters (Millipore
Corp., Bradford, MA), probed with peroxidase-conjugated streptavidin,
and visualized on Kodak X-OMAT AR films (Rochester, NY) by the Enhanced Chemiluminescence System (Amersham Life Sciences, Little
Chalfont, UK). In biotinylation experiments of
1 integrins, cells were
previously treated with 100 µg/ml trypsin in PBS twice for 20 min at 4°C,
followed by inactivation with complete medium (Boll et al., 1991
).
Adhesion Assay
Cell adhesion was performed according to Grano et al. (1994), with minor
modifications. In brief, 96-well microtiter plates (polystyrene, nontissue
culture treated; Nunc Inc., Naperville, IL) were coated with increasing
concentrations of vitronectin (VN; Sigma Chemical Co.), fibrinogen
(Sigma Chemical Co.), and osteopontin (from Cecilia M. Giachelli and
Marta Scatena, Department of Pathology, University of Washington, Seattle, WA) in PBS, pH 7.4. In other assays, standard concentrations of 10 µg/ml laminin (Sigma Chemical Co) and fibronectin (FN; from Paola Defilippi, University of Torino) were used. Proteins were allowed to bind overnight at 4°C before the wells were rinsed and blocked for 2 h at 37°C
with 3% heat-denatured BSA (RIA grade; Sigma Chemical Co.) in PBS,
pH 7.4. Cells were harvested and washed twice with serum-free medium
(SFM). To allow surface reexposure of integrin receptors, cells were incubated on a rotating platform for 1 h at 37°C in SFM containing 0.1% BSA,
and then added to the wells at a concentration of 50,000 cells/0.1 ml of the
same medium. After a 3-h incubation at 37°C, wells were gently washed
twice in PBS. Adherent cells were fixed in 11% glutaraldehyde in PBS, rinsed in distilled water, and stained with 0.1% crystal violet, 20% methanol for 15 min. Cell numbers were obtained by counting all cells in four
grids using a phase-contrast light microscope fitted with a 32 grid eyepiece
at a total magnification of 100× (Doerr and Jones, 1996
). All data presented are the means ± SD of duplicate wells from three or more experiments. Nonspecific cell adhesion as measured on BSA-coated wells has
been subtracted.
In adhesion inhibition experiments, cells were plated onto the substrata in the presence of serial dilutions of the function-blocking mAbs LM609 or AIIB2. Alternatively, HTU-34 cells were treated for 2 d with 250 µg/ml suramin or with 1W53 antibody against HGF/SF and then processed for the adhesion assay; suramin or the inhibitory antibody was kept in all steps of the assays. Preimmune sera were used in control experiments.
In some assays, adherent HTU-5 cells were serum-starved for 36 h, harvested, and plated onto VN in SFM-0.1% BSA in the presence of single
GFs or HTU-34-conditioned medium. In the case of TGF1 treatment,
before harvesting cells were pretreated with TGF
1 for 24 h. Alternatively, HTU-34-conditioned medium was preincubated with the 1W53 antibody against HGF/SF or with sheep normal IgGs (Sigma Chemical Co.)
for 30 min and then applied to HTU-5 cells (see Results).
Indirect Immunofluorescence Microscopy
Cells from confluent monolayers were plated onto 24-well plates (Costar
Corp.) containing 1.4-cm2 glass coverslips. After 4 d in culture, cells were
fixed for 5 min at room temperature in a freshly prepared solution of 3% formaldehyde (from paraformaldehyde) in PBS, pH 7.6, containing 2%
sucrose. In some cases, cells were permeabilized by soaking coverslips for
3 min at room temperature in Hepes-Triton X-100 buffer (20 mM Hepes,
pH 7.4, 300 mM sucrose, 50 mM NaCl, 3 mM MgCl2, and 0.5% Triton X-100;
Fey et al., 1983; Rabinovitz and Mercurio, 1997
). Indirect immunofluorescence was performed as previously reported (De Luca et al., 1990
;
Marchisio et al., 1991
). In brief, after a 15-min saturation with PBS-2%
BSA at 37°C, the primary antibodies were layered onto cells and incubated in a moist chamber for 30 min. After rinsing in PBS-0.2% BSA, coverslips were incubated with the appropriate rhodamine-tagged secondary antibody (DAKOPATTS, Copenhagen, Denmark) for 30 min at 37°C in the presence of 2 µg/ml of fluorescein-labeled phalloidin (Sigma Chemical Co.). Coverslips were mounted in Mowiol 4-88 (Hoechst AG, Frankfurt, Germany) and observed in a photomicroscope (Axiophot; Zeiss,
Jena, Germany) equipped with epifluorescence lamp and planapochromatic oil immersion lenses. Fluorescence images were recorded on Kodak T-Max 400 photographic films exposed at 1,000 ISO and developed in
T-Max Developer for 10 min at 20°C.
In some experiments, coverslip-attached HTU-5 cells were serum-starved for 36 h and then treated with HGF/SF or with HTU-34-conditioned medium (see Results). In other cases, HTU-34 cells were treated for 2 d with 1W53 antibody against HGF/SF or with sheep preimmune sera (Sigma Chemical Co.) and then processed for immunofluorescence.
FACS® Analysis
FACS® analysis was performed according to Peruzzi et al. (1996), with minor modifications. In brief, HTU-5 and HTU-34 cells were harvested with
1 mM EDTA in PBS, washed in ice-cold PBS-2%BSA, 5 mM NaN3, and
incubated with mAb VIPL-2 against the
3 integrin subunit (10 µg/ml) for
40 min at 4°C. Cells were then rinsed and treated with fluorescein-tagged
rabbit anti-mouse IgG (DAKOPATTS) for 30 min. All incubations were
performed in PBS-0.2%BSA, 5 mM NaN3 at 4°C. Fluorescence was measured using a FACScan® flow cytometer (Becton Dickinson, Mountain
View, CA) set to count 10,000 cells per sample. The data were collected
and analyzed with a MacIntosh Power PC computer equipped with
CELLQuest research software (Becton Dickinson). Positive fluorescence
was determined on a four log scale and expressed as channel number
mean intensity fluorescence (MIF). Background fluorescence was determined on each cell population using fluorescein-tagged rabbit anti-mouse IgGs alone.
Northern Blotting
Total RNA was isolated from confluent monolayers of HTU-5 and HTU-34 cells by the acid guanidium method (Chomczynski and Sacchi, 1987).
Northern blots were performed with 30 µg total RNA per lane. Ethidium
bromide at a concentration of 0.2 µg/ml was added before electrophoresis
in 1% agarose gels containing formaldehyde to verify the integrity of the
RNA by short-wavelength UV detection and to monitor the equivalence
of loading before and after transfer to GeneScreen Plus filters (Du Pont
NEN, Boston, MA). A full-length HGF/SF cDNA probe (from Gianni
Gaudino, Dipartimento di Scienze e Tecnologie Avanzate, University of
Alessandria, Italy) and a probe for the housekeeper gene glyceraldehyde-3
phosphate dehydrogenase (GAPDH, from Fanny Sciacca, DIBIT, Milano, Italy) were labeled with random priming (Rediprime DNA labeling
system; Amersham Life Sciences) and [32P]dCTP (3,000 Ci/mmol; Amersham Life Sciences). Membranes were pretreated and hybridized in 50%
formamide (Merck, Darmstad, Germany), 10% dextran sulfate (Sigma
Chemical Co.), 1% SDS, and 50 µg/ml salmon sperm DNA, at 42°C. Blots
were washed twice with 2× SSC at room temperature for 10 min, then
twice in 2× SSC 1% SDS at 65°C for 30 min, and finally once in 2× SSC at
room temperature for 5 min, followed by exposure to autoradiography for
48 h at -80°C with intensifying screens.
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Results |
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Thyroid Clonal Strains Express v
3,
v
1, and
3
1 Integrins
To characterize the surface adhesive repertoire of normal
(HTU-5) and malignant (HTU-34) thyroid cells, a battery
of integrin-specific mAbs was used in immunoprecipitation experiments on membrane biotinylated cell monolayers. The v mAb L230 immunoprecipitated three bands of
150, 130, and 90 kD in both the normal and carcinoma
strains (Fig. 1 A). The 150/90-kD doublet was also brought
down by the
3-specific mAb VIPL2 (Fig. 1 B) but not by
the
5 mAb IA9 (not shown). Based on band intensities,
the
v
3 integrin appeared to be more expressed in the
HTU-34 clone. The higher surface exposure of
v
3 in
carcinoma cells was confirmed by FACS® analysis on
HTU-5 (Fig. 1 C) and HTU-34 (Fig. 1 D) cells.
|
The faint 130-kD band coprecipitating with the v subunit could be interpreted as an
v-associated
1 chain. To
test the nature of this band, anti-
v immunoprecipitates
from equal amounts of HTU-5 and HTU-34 cell extracts
were transferred onto Immobilon-PTM membranes and
probed with a
1 polyclonal antiserum: in fact, a specific
130-kD band corresponding to the
1 subunit was detected in both clones (Fig. 1 E). Under standard conditions, surface biotinylation of
1 integrins was extremely
difficult, possibly because the accessibility of this integrin
to biotin was compromised by the adhesive meshwork of
basement membrane components (Gottardi and Caplan,
1992
). To overcome this problem we employed a mild trypsinization protocol that enhances the ability of biotin
to interact with ventral proteins (Boll et al., 1991
). By this
procedure, immunoprecipitation of HTU-5 and HTU-34
cell lysates with the
1 mAb MAR4 yielded two bands of
similar intensity at 150 and 130 kD, representing one or
more
1-associated
chains and the
1 subunit (Fig. 1 F),
respectively. To further define which
subunits, besides
v, could heterodimerize with the
1 chain, mAbs against
2,
3,
5, and
6 were used in immunoprecipitation assays on biotinylated normal and malignant cells. Only the
3-mAb F2 was able to precipitate two bands of 150 and
130 kD comigrating with the
3 and
1 integrin subunits
(Fig. 1 G). These same mAbs were also used in immunofluorescence experiments; consistently with the immunoprecipitation analysis, among the
subunits tested only
3
and
v were immunoreactive (not shown).
Taken together, these data demonstrate that the integrins expressed at the surface of normal and malignant
thyroid clones include the v
1,
v
3, and
3
1 heterodimers. The
1 chain is thus shared by the
3 and
v
subunits.
The Integrin v
3 Is Clustered at Focal
Contacts and Mediates Adhesion in Malignant but Not
in Normal Thyroid Cells
The only modification in integrin repertoire observed in
malignant versus normal thyroid cells was the higher surface expression of the v
3 heterodimer in HTU-34 cells
(Fig. 1, A-D). To evaluate the subcellular distribution of
v
3 in normal and carcinoma clones, cells were plated
onto glass coverslips, cultured for several days, and then
subjected to immunofluorescence. Under these conditions, cell adhesion occurs because of endogenous production of matrix molecules. Immunofluorescence experiments on fixed, nonpermeabilized cells were in accordance
with the immunoprecipitation data: a fine grainy pattern
of immunoreactivity was much stronger in HTU-34 (Fig. 2
B) than in HTU-5 cells (Fig. 2 A). Interestingly, treatment of fixed cells with permeabilization buffer (0.5% Triton
X-100), which extracts freely diffusing molecules yet preserves actin cytoskeletal connections (Fey et al., 1983
;
Rabinovitz and Mercurio, 1997
), completely removed
3
immunoreactivity in HTU-5 cells (Fig. 2, C and D) and selectively concentrated the
3 fluorescent signal of HTU-34
cells at the endings of microfilament bundles in sites compatible with FCs (Fig. 2, E and F). The same result was obtained when Triton X-100 permeabilization was performed before fixation (not shown). Thus, recruitment of
the
v
3 heterodimer to microfilament-associated adhesion sites occurs only in malignant but not in normal thyroid cells.
|
To induce ligand-mediated clustering of v
3 in HTU-5
normal cells, subconfluent cultures were detached and
plated in serum-free conditions onto a plastic substratum
coated with a concentration range (2.5 to 25 µg/ml) of VN
(Fig. 3 A), fibrinogen (Fig. 3 B), and osteopontin (Fig. 3
C). Surprisingly, HTU-5 cells could not attach and spread. In
contrast, HTU-34 cells rapidly adhered and firmly spread;
indeed, cells attached proportionally to the amount of substratum and were detectable even at very low doses of matrix ligands (Fig. 3, A-C). HTU-34 cell attachment and
spreading on VN was progressively impaired by adding increasing concentrations of the
v
3 inhibitory mAb
LM609 but not by the
1 function-blocking mAb AIIB2
(Fig. 3 D). Moreover, in HTU-34 cells plated on VN and processed for immunofluorescence after fixation and permeabilization, the only integrin receptor clustered at nascent FC, strictly colocalizing with vinculin (Fig. 3 F), was
v
3 (Fig. 3 E). Conversely,
1 integrins were almost undetectable on the cell surface (Fig. 3 G). Thus, adhesion of
HTU-34 cells to VN was specifically mediated by
v
3.
|
When HTU-5 were plated onto FN, a ligand for both
v
3 and
v
1, cells could attach and spread. In this case
as well,
v
3 was not involved in the adhesive phenomenon: only the
1 inhibitory mAb efficiently blocked adhesion whereas mAb LM609 did not display any significant
effect (Fig. 4 A). Conversely,
1 and
3 integrins were
equally responsible for adhesion to FN in HTU-34 cells:
function-blocking mAbs against either integrins could partially impair adhesion when added individually, and almost totally when added together (Fig. 4 A). Immunofluorescence experiments showed that HTU-5 cells, when
plated on FN, organized
1 integrins at adhesive structures (Fig. 4 B), whereas
v
3 was almost undetectable
(Fig. 4 C). On the contrary, in HTU-34 plated on FN both
1 and
3 integrins were highly enriched at focal adhesions; double immunostaining for
1 and
3 revealed colocalization of the two integrin subunits within the same FCs
(Fig. 4, D and E).
|
In summary, these results indicate that adhesion to VN,
fibrinogen, and osteopontin occurs only in malignant
HTU-34 cells and that, in this strain, adhesion is specifically driven by v
3. Adhesion to FN occurs in both
strains and is governed by the selective activity of
1 integrins in HTU-5 cells and by the cooperative action of
1
and
3 integrins in HTU-34 cells.
We hypothesized that the discrepancy in adhesion efficiency between HTU-5 and HTU-34 cells was due to the
different expression levels of the v
3 integrin receptor.
To test this hypothesis, HTU-5 were treated with TGF-
1,
known to induce upregulation of
v
3 synthesis and surface exposure (Ignotz et al., 1989
). Indeed, TGF-
1 increased the expression of
v
3 but could not induce
v
3 recruitment at cytoskeleton-associated FCs nor enhance
HTU-5 cell adhesion to VN (not shown). These findings
suggest that
v
3 upregulation is not sufficient per se to
trigger cluster formation and firm adhesion in HTU-5
cells. Thus, we can reasonably rule out the possibility that
the different adhesive capabilities of carcinoma versus
normal cells are simply related to the higher surface exposure of
v
3 in the HTU-34 clone.
A Carcinoma-specific Autocrine Loop Sustains
v
3-mediated Adhesion
Since the assembly of integrin adhesion complexes requires serum soluble factors in some cell types (Hotchin
and Hall, 1995), and on the basis of mounting evidence
that integrins and GF receptors share common signaling
pathways (Clark and Brugge, 1995
), we assumed that signals derived from a GF receptor could be responsible for maintaining
v
3 in a constitutively proadhesive activated
state.
In a preliminary test of this possibility we plated HTU-34
cells on VN after preincubation with suramin, a drug that
blocks any cytokine-receptor interactions (La Rocca et al.,
1990; Adams et al., 1991
; Ferracini et al., 1995
; Zumkeller
and Schofield, 1995
). Indeed, HTU-34 cells completely
lost their adhesion potential (Fig. 5 A) suggesting that
v
3 adhesive properties were controlled by a soluble factor interacting with a receptor. To test this hypothesis we
challenged HTU-5 cells with SFM conditioned by the HTU-34 clone and found that cells acquired de novo adhesion to VN (Fig. 5 A). When HTU-34-conditioned medium was applied to HTU-5 cells previously plated onto
glass coverslips, thus adhering to endogenous ECM molecules,
v
3 recruitment at FCs was observed (Fig. 5, B and
C). It was deduced that a soluble factor produced by malignant cells, but not by normal cells, controlled
v
3-mediated adhesion by acting on a receptor shared by the
two cell types.
|
HGF/SF Promotes v
3-mediated Adhesion
The multifunctional cytokine HGF/SF was selected as the
object of closer investigation for several reasons: (a) the
HGF/SF receptor c-Met is constitutively activated in thyroid carcinomas (Di Renzo et al., 1992, 1995
); (b) HGF/SF
autocrine release has been reported to represent a selective advantage for tumor progression (Tsao et al., 1993
;
Ferracini et al., 1995
); (c) the morphogenic responses to
HGF/SF are critically dependent on cell adhesion (Matsumoto et al., 1995
); and (d) finally, HGF/SF has been shown to enhance adhesion of B cells and lymphoma cells, thus
suggesting its involvement in integrin activation mechanisms (Van der Voort et al., 1997
; Weimar et al., 1997
).
Indeed, HGF/SF clearly promoted attachment and
spreading of HTU-5 cells on VN in a dose-dependent
manner (Fig. 6 A). HGF/SF-induced adhesion was specifically inhibited by mAb LM609 against v
3 (Fig. 6 B). In
agreement with these findings, HTU-34 adhesion on VN
was impaired by a functional antibody to HGF/SF (Fig. 6 B). Moreover, this antibody, but not normal sheep serum,
blocked the ability of HTU-34-conditioned medium to induce adhesion of HTU-5 cells (Fig. 6 B). The proadhesive
effect of HGF/SF was specific insofar that TGF-
1 could
not enhance HTU-5 cell adhesion to VN (not shown) nor
could EGF, insulin, and insulin-like growth factor-1 (Fig. 6
B). It has already been demonstrated that receptors for EGF, insulin, and insulin-like growth factor-1 are present
in thyroid cells (Dumont et al., 1991
); however, to ascertain that also HTU-5 cells express these receptors, we performed Western blot experiments on total cell lysates after
GF stimulation and verified the induction of multiple tyrosine phosphorylated bands (not shown).
|
When HTU-5 cells were plated on FN in the presence of
HGF/SF, adhesion was markedly enhanced (Fig. 6 C).
Blockade of the 1 integrin receptors by means of mAb
AIIB2 under basal conditions abolished adhesion, that
was partially restored by adding HGF/SF (Fig. 6 C). Adhesion levels after inhibition of
v
3 by mAb LM609 in the
presence of HGF/SF were roughly comparable to those
obtained under basal conditions (Fig. 6 C). When both
1
and
3 integrins were blocked by their respective inhibitory mAbs, adhesion was totally abolished and stimulation
with HGF/SF was ineffective (Fig. 6 C). Taken together,
these results indicate that HGF/SF enhances adhesion efficiency of HTU-5 cells on FN by promoting
v
3-mediated
attachment and spreading. Hence,
v
3 activation significantly increases the level of basal adhesion mediated by
1
integrins.
As a control, HTU-5 and HTU-34 cells were plated on
laminin (Fig. 6 D). Both clones adhered at comparable levels and adhesion was blocked by the 1 mAb AIIB2, acting conceivably on the
3
1 heterodimer. Addition of
HGF/SF did not modify cell attachment and spreading. Thus,
the effect of HGF/SF on cell adhesion is specifically mediated by
v
3 and is independent of the substratum recognized by this integrin, being exerted on both VN and FN.
In immunofluorescence experiments on HTU-5 cells adhering onto endogenous ECM molecules, HGF/SF triggered clustering of v
3 at FCs (Fig. 7, A and B). Consistent with this observation, treatment of HTU-34 cells with
the inhibitory antibody to HGF/SF resulted in a clear-cut
decrease in the focal immunostaining for
v
3 (Fig. 7, C
and D), but no modifications in the topography of the
1 subunit (Fig. 7, E and F) or vinculin (Fig. 7, G and H) were
observed.
v
3 expression levels in HTU-5 clones did not
increase upon stimulation with HGF/SF, nor did they decrease after antibody-mediated neutralization of HGF/SF
activity in the HTU-34 cultures (not shown).
|
To determine whether HTU-5 and HTU-34 cells expressed the HGF/SF receptor c-Met and to verify whether
the receptor was constitutively activated in HTU-34 cells
because of a chronic autocrine loop, cell lysates were subjected to immunoprecipitation with the C-28 human Met
polyclonal antibody. Anti-Met immunoprecipitates were then split into two equal fractions, Western blotted, and
decorated with the anti-Met mAb DQ-13 (Fig. 8 A) or the
phosphotyrosine mAb 4G10 (Fig. 8 B). In both cell lines,
the 145-kD mature form of the c-Met subunit was clearly
detected (Fig. 8 A). The c-Met
subunit was phosphorylated on tyrosine residues in HTU-34 cell extracts, but not
in unstimulated HTU-5 cell lysates. When HTU-5 cells
were treated with conditioned medium from the HTU-34
clone or with purified HGF/SF, specific tyrosine phosphorylation of the c-Met
subunit was detected (Fig. 8 B).
|
The presence of HGF/SF in the supernatant of HTU-34 cells was tested by assaying its scatter activity in MDCK epithelial cells after serial dilutions in standard medium. At a 1:10 dilution, conditioned supernatant was able to dissociate epithelial colonies, although it did not achieve the maximal effect observed with recombinant HGF/SF (not shown).
Finally, Northern blot analysis identified a specific 6-kb
transcript, equivalent in size to the principal HGF/SF
mRNA species (Nakamura et al., 1989), only in HTU-34
cells (Fig. 8 C). We believe that these results unequivocally substantiate the existence of a natural autocrine loop
for HGF/SF in HTU-34 cells.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
A New Physiological Mechanism for Integrin Activation in Epithelia
Normal epithelial cells require integrin-mediated adhesion
to ECM molecules of the basement membrane for GF
control of the cell cycle (Assoian, 1997). In this paper we
report that the opposite phenomenon also occurs, i.e., that
integrin-dependent cellular adhesion requires GFs to take
place. Namely, we report that the HGF/SF-dependent signal transduction pathway can induce ligand-binding activity in functionally inactive
v
3 integrins.
The activation of integrins is characterized by conformational changes in their extracellular domain, reorganization of their cytoplasmic connections, and clustering of
heterodimers within the plane of the plasma membrane,
which together augment integrin affinity and avidity for
ligands and stabilize adhesion (Du et al., 1991; Diamond
and Springer, 1994
; Li et al., 1995
; Yednock et al., 1995
).
The molecular mechanisms responsible for physiological activation are still unclear (Lasky, 1997
). Moreover, although this phenomenon has been extensively studied in
platelets and leukocytes, little information is available for
cells that are part of compact tissues and adhere to basement membranes, such as epithelial cells. Recently, Pelletier et al. (1996)
reported that
v
3 activation in a melanoma cell line involves a cation binding site that regulates
integrin conformation. Even more recently, Fenczik et al.
(1997)
identified CD98, a type II membrane glycoprotein involved in early T cell activation and expressed in many
adherent cell lines, as a factor responsible for
1 integrin
activation. The mechanisms that regulate clustering of integrins and their recruitment at FCs also are poorly understood. Experiments with antibody- and ligand-coated
beads have shown that clustering of integrins depends
upon binding to multivalent matrix molecules and that FC
assembly requires both integrin-ligand interaction and aggregation of integrins (Miyamoto et al., 1995
).
Data presented in this study indicate that both integrin
aggregation and triggering of efficient ligand-binding capability in adherent normal cells require the presence of
GFs; in particular, HGF/SF displays the unique ability to
recruit v
3 to FCs and to stimulate
v
3-mediated adhesion. Since no changes in
v
3 expression levels can be
observed upon HGF/SF treatment, we interpret HGF/SF-induced adhesion as a conversion of the integrin functional state from inactive to active, with consequent acquisition of ligand-binding ability. To our knowledge, these
findings are the first example of a GF-driven integrin activation mechanism in adherent cells. Moreover, because
we use an epithelial cell model presumably mirroring the
adhesive environment of solid normal tissues and tumors,
we elucidate one of the mechanisms that coordinate integrin and GF receptor function in normal and transformed
epithelia under conditions that parallel several in vivo situations.
How can HGF/SF elicit clustering and activation of
v
3? One possibility is that the activated GF receptor directly acts on the
3 cytoplasmic domain and that this, in
turn, induces a conformational change in the integrin resulting in ligand binding. Integrin-ligand interaction
would then trigger
v
3 clustering at FCs. However, we
could never show any obvious biochemical modification of
the
3 integrin tail like HGF/SF-induced tyrosine phosphorylation nor formation of a complex between the activated HGF/SF receptor and the
v
3 integrin.
A second possibility is that HGF/SF activates the v
3
integrin in an indirect manner, e.g., by modifying a range
of intermediate effectors. Good candidates for this role
would include CD98 analogues (see above) or
3-endonexin, a cytosolic protein that selectively binds the
3
cytodomain and modulates its affinity state (Shattil et al.,
1995
; Kashiwagi et al., 1997
).
In a third scenario, HGF/SF might affect one or more of
the submembranous components of the FCs. Because
most of these components, as well as integrin cytoplasmic
tails, have been shown to interact in vitro with each other,
this could lead to v
3 recruitment via the formation of
multiple cross-links (Turner and Burridge, 1991
; Sastry
and Horwitz, 1993
; Gilmore and Burridge, 1996
). According to this hypothesis, clustered integrins would form a
high density, high valency complex with increased avidity
for ligands, thus leading to stabilization of integrin-ligand
interactions, firm adhesion, and spreading. Interestingly,
HGF/SF is known to induce tyrosine phosphorylation of
pp125FAK (Matsumoto et al., 1994
), a cytosolic tyrosine kinase enriched at FCs and able to phosphorylate other FC
components including paxillin and tensin (Schaller and
Parsons, 1994
).
Implications for Tumor Invasion
All the morphological and functional features evoked by
HGF/SF in normal thyroid cells spontaneously occur in
carcinoma cells because of a natural HGF/SF autocrine
loop. Inhibition of this loop markedly reduces v
3 enrichment at FCs and binding to immobilized ligands.
Hence, whereas HGF/SF treatment of normal cells recapitulates the overall adhesive phenotype of carcinoma
cells, neutralization of HGF/SF activity in neoplastic elements can per se revert
v
3 from a functional to a latent
state.
Although in different ways, both HGF/SF and v
3 integrin contribute to the malignant behavior of neoplastic
cells. HGF/SF is responsible for invasive growth of tumors, a complex phenomenon resulting from the combination of proliferation, motility, ECM degradation, and cell
survival. Specifically, HGF/SF impairs the compaction of
polarized epithelia by disrupting the architecture of adherens junctions and inducing the appearance of a fibroblastoid phenotype endowed with motile properties (Stoker et
al., 1987
; Gherardi et al., 1989
; Weidner et al., 1990
, 1991
;
Igawa et al., 1991
; Kan et al., 1991
; Matsumoto et al., 1991
;
Naldini et al., 1991
; Rubin et al., 1991
). This scatter activity
is corroborated by HGF/SF ability to promote the synthesis of ECM-degrading proteases, including urokinase plasminogen activator (uPA; Pepper et al., 1992
; Boccaccio et al.,
1994
; Jeffers et al., 1996
) and matrix metalloproteinase-2
(MMP-2; Zeigler et al., 1996
), thus enhancing cell invasiveness into stromal compartments. Finally, HGF/SF can protect epithelial cells from anoikis, a form of programmed cell death occurring when adherent cells are detached from their physiological matrix substrata (Frisch
and Francis, 1994
; Longati et al., 1996
; Amicone et al.,
1997
). In fact, when carcinoma cells infiltrate connective
tissues and blood vessels before systemic dissemination, they lose contact with their basal lamina and, to escape
anoikis, must recognize previously unknown ECM components. It is tempting to speculate that the survival message
conveyed by HGF/SF resides, at least partially, in its ability to activate the function of the
v
3 integrin, thus supplying an adhesive information that may confer resistance
to anoikis. From this viewpoint, the ability of HGF/SF to
activate
v
3 in carcinoma cells results in a double selective advantage: (a) it provides a functional receptor for stromal invasion and (b) it protects tumors from massive
apoptosis.
Similarly to HGF/SF, v
3 is directly involved in invasive and antiapoptotic phenomena. This integrin is upregulated in melanoma clones endowed with high metastatic
potential (Nip et al., 1995
) and is physically associated
with MMP-2 at the invasive front of infiltrating cells, in order to concentrate matrix degradation at adhesive sites
and facilitate directed cellular motility (Brooks et al.,
1996
). Interestingly,
v
3 is de novo expressed on the surface of endothelial cells during intratumoral formation of blood vessels (Brooks et al., 1994
) and regulates the survival of newly sprouting blood vessels (Stromblad et al.,
1996
); likewise, HGF/SF displays powerful angiogenic activity (Bussolino et al., 1992
). Altogether, data support the
concept of a functional synergy between HGF/SF-dependent biological pathways and
v
3-mediated adhesion
processes in several neoplastic phenomena including matrix degradation, invasion, cell survival, and tumor neoangiogenesis.
It is worth noting that in many cell lines (the HTU-5 thyroid clone being a prominent exception) v
3 appears to
be spontaneously clustered at FCs. In some cells, the basal
activation state of the integrin may be intrinsically high,
or perhaps more likely, maintained by autocrine production of HGF/SF. Accordingly, many examples of natural
autocrine cells for HGF/SF have been described (Adams et
al., 1991
; Rong et al., 1992
, 1993
; Tsao et al., 1993
; Grano et
al., 1994
; Ferracini et al., 1995
; Woolf et al., 1995
; Maier
et al., 1996
; Anastasi et al., 1997
). An alternative hypothesis stems from the observation that in a variety of cell lines
simple cellular adhesion is sufficient to elicit ligand-independent activation of the HGF/SF receptor (Wang et al.,
1996
). In this case, recruitment of
v
3 at FCs would be
the consequence of adhesion-dependent constitutive activation of the kinase receptor rather than a cellular response to chronic autocrine stimulation by the growth factor ligand. Notably, activation of the HGF/SF receptor by
cell attachment occurs in many tumor cells, but not in primary or clonal cultures of normal cells (Wang et al., 1996
).
In conclusion, we propose here a novel regulatory mechanism that epithelial cells use for integrin activation and in
the ensuing integrin-ligand interaction phenomena. To
our knowledge, this is the first report describing the specific modulation operated by a GF on the adhesive state
and aggregation rate of an individual integrin in epithelial
cells. Moreover, because v
3 activation is obtained upon
stimulation with a GF that is physiologically present
within the interstitial milieu of compact tissues and can be
pathologically overexpressed in cancer, this mechanism can have strong in vivo implications for the adhesive behavior of parenchymal cells and for their interactions with
stromal components. In addition, our results highlight the
importance of GF autocrine production in the regulation
of integrin function during tumor invasion.
![]() |
Footnotes |
---|
Received for publication 4 February 1998 and in revised form 15 July 1998.
The major support for this work was from Associazione Italiana per la Ricerca sul Cancro (AIRC, Milano, Italy) to PCM. Partial support came from Agenzia Spaziale Italiana (ASI, Roma, Italy) to PCM within a space biology program aimed at studying cell adhesion in ground experiments.We gratefully acknowledge our colleagues for the gifts of reagents described in Materials and Methods. In particular, we are indebted to Paolo Amati and Sergio Anastasi for providing the inhibitory antiserum to HGF/SF. We thank Robert B. Low, Stefano J. Mandriota, and Antonio Pinto for critically reading this manuscript. Thanks also to Fabrizio Guidi for help with the FACS® analysis and to Enrico Saggiorato for assistance with the photographic work. Filomena Ciarfaglia and Silvana Costa provided excellent technical assistance.
![]() |
Abbreviations used in this paper |
---|
ECM, extracellular matrix; FC, focal contact; FN, fibronectin; GF, growth factor; HGF/SF, hepatocyte growth factor/scatter factor; MIF, mean intensity fluorescence; MMP-2, metalloproteinase-2; SFM, serum-free medium; VN, vitronectin.
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