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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 8 (October 15), 1999:
pp. 2754-2766
Fibronectin Upregulates Gelatinase B (MMP-9) and Induces Coordinated
Expression of Gelatinase A (MMP-2) and Its Activator MT1-MMP
(MMP-14) by Human T Lymphocyte Cell Lines. A Process Repressed
Through RAS/MAP Kinase Signaling Pathways
By
Jordi Esparza,
Carme Vilardell,
Javier Calvo,
Manel Juan,
Jordi Vives,
Alvaro Urbano-Márquez,
Jordi Yagüe, and
Maria C. Cid
From the Departments of Internal Medicine and Immunology, Hospital
Clínic, University of Barcelona, IDIBAPS (Institut d'
Investigacions Biomèdiques August Pi i Sunyer), Barcelona, Spain;
and Immunology Unit, Hospital Germans Trias i Pujol, Badalona, Spain.
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ABSTRACT |
T-lymphocyte migration into tissues requires focal degradation of
the basement membrane. In this study, we show that transient adherence
to fibronectin induces the production of activated forms of matrix
metalloproteinase-2 (MMP-2) and MMP-9, as well as downregulation of
tissue inhibitor of metalloproteinase-2 (TIMP-2) by T-cell lines. MMP-2
activation was likely achieved by inducing a coordinated expression of
membrane-type matrix metalloproteinase-1 (MMP-14), a major activator of
MMP-2. Blocking monoclonal antibodies against  4, 5, and v
integrins strongly reduced MMP-2 and MMP-9 production induced by
fibronectin. Disrupting actin cytoskeleton organization by cytochalasin
D strongly enhanced fibronectin-induced MMP-2 and MMP-9 expression.
Inhibiting Src tyrosine kinases with herbimycin A reduced MMP-2 and
MMP-9 production with no effect on cell attachment. By contrast,
G-protein inhibition by pertussis toxin, or transfection with a
dominant negative mutant of Ha-Ras strongly increased
fibronectin-induced MMP-2 and MMP-9. Inhibition of PI3 kinase,
MAPkinase (MEK1), or p38 MAPkinase by wortmannin, PD 98059, or SB
202190, respectively, strongly promoted fibronectin-induced MMP2 and
MMP-9. Cells at high density lost their ability to synthesize MMP-2 and
MMP-9 in response to fibronectin and MMP expression was restored by transfection with a dominant-negative mutant of Ha-Ras or by treatment with wortmannin, PD 98059, or SB 202190. Our findings suggest that
adhesion to fibronectin transduces both stimulatory (through Src-type
tyrosin kinases) and inhibitory signals (through Ras/MAPKinase signaling pathways) for MMP-2 and MMP-9 expression by T lymphocytes and
that their relative predominance is regulated by additional stimuli
related to cell adhesion, motility, and growth.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
THE DEVELOPMENT OF inflammatory
infiltrates in target tissues is a common pathologic substrate in many
chronic inflammatory and autoimmune diseases.1 Tissue
infiltration by T lymphocytes requires dynamic and finely regulated
interactions between lymphocytes, endothelial cells, and the underlying
basement membrane mediated via a complex array of surface
receptors.2-4 Lymphocyte receptors participating in the
interactions with endothelial cells belong to two major classes of
proteins: selectins that mediate the very initial interactions with
carbohydrate ligands on the endothelial cell surface, and integrins
that are responsible for the tight adhesion and further transmigration
through the endothelial cell junctions.
Once the transmigration process is accomplished, lymphocytes begin to
interact with the underlying basement membrane and interstitial matrix.5-7 The basement membrane is a specialized
extracellular matrix structure synthesized by epithelial and
endothelial cells composed essentially of collagen type IV, laminin,
and perlecan. Additional important components are nidogen/entactin,
collagen type V, and fibronectin among other glycoproteins and
proteoglycans.8 Its structural organization in multilayers
conforms a resistant barrier.
Vectorial motility across the basement membrane and interstitial matrix
requires coordinated series of adhesion-release steps and focal matrix
degradation.7,9,10 Lymphocyte integrins have a major role
in mediating interactions with extracellular matrix proteins during the
transmigration process.5,7,9-11 However, the mechanisms
involved in basement membrane degradation by transmigrating lymphocytes
are much less understood. Recent contributions suggest an important
role for matrix metalloproteinases in basement membrane disruption by T
lymphocytes.12
The matrix metalloproteinase (MMP) family includes a growing family of
endopeptidases that have been classified according to their substrate
specificity into gelatinases, stromelysins, and
collagenases.10,13,14 Recently, a new subclass of
transmembrane MMP (MT-MMP) expressed on the surface of invasive tumor
cells or in the surrounding stromal cells has been
identified.14 MMP are synthesized in an inactive form or
zymogen with an aminoterminal propeptide that must be cleaved to yield
the active enzyme.10,13,15
The 92 kD gelatinase (gelatinase B, MMP-9) and the 72 kD gelatinase
(gelatinase A, MMP-2), efficiently degrade native collagen types IV and
V, fibronectin, entactin, and elastin. Therefore, these proteases are
believed to be of crucial importance in processes requiring basement
membrane disruption such as tumor invasion and
metastasis10,13-16 and, presumably, tissue infiltration by leukocytes, the pathologic substrate of chronic inflammatory diseases. Gelatinases are secreted in association with specific inhibitors of MMP
or TIMP (MMP-2/TIMP-2 and MMP-9/TIMP-1) that bind to the carboxy-terminal domain of progelatinases.10,13,15,17
The precise mechanisms of gelatinase activation are beginning to be
understood. Other members of the MMP family have been proposed as
putative physiologic activators of progelatinases.18 Recently, membrane-bound MMP MT1-MMP (MMP-14) has been shown to be a
potent activator of progelatinase A by cleaving the propeptide, a
process that also promotes gelatinase-A autoproteolytic
activation.14,19 Gelatinase A, in turn, is able to process
interstitial collagenase (MMP-1)20 and collagenase-3
(MMP-13).21
It has been recently shown that T lymphocytes constitutively produce
small amounts of gelatinase B (MMP-9), which is highly upregulated upon
phorbol ester22 or interleukin-2 (IL-2)
stimulation.22 T- and B-lymphoblastoid cell lines also
exhibit baseline production of MMP-9.24 In T-lymphoblastoid
cell lines chemokines, such as MIP-1 and RANTES, and cytokines, such
as IL-2 and IL-4, upregulate MMP-9 expression.25 Functional
studies have shown that increased MMP-9 production leads to an enhanced
invasiveness through reconstituted basement membrane
Matrigel.23,25 Gelatinase-A (MMP-2) production by T
lymphocytes has only been detected under much more restricted conditions such as IL-2 stimulation for long periods of
time23 or upon VCAM-1-dependent adhesion to cultured
endothelial cells.26 However, the mechanisms underlying MMP
expression and activation by T lymphocytes have not been investigated.
According to the hypothesis that attachment to the extracellular matrix
proteins and their subsequent degradation may be related events, the
purpose of our study was to determine whether basement membrane
components are able to modulate MMP synthesis by human lymphocytes.
Herein, we show that, among several basement membrane constituents,
only intact fibronectin possesses the information necessary not only
for inducing MMP-9 and MMP-2 production by T-cell lines but also for
promoting MMP-2 activation, probably by inducing coordinated expression
of MT1-MMP.
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MATERIALS AND METHODS |
Monclonal antibodies (MoAb), chemicals, extracellular matrix proteins,
and peptides.
Blocking MoAb anti- 1 chain (CD29) K20 and anti-5 chain (CD49e)
SAM1 were purchased from Immunotech (Marseille, France). Blocking MoAb
anti-4 (CD49d) HP 2/1 and anti-v (CD51) 17E6 were kindly provided
by Dr F. Sánchez-Madrid (Hospital La Princesa, Madrid, Spain) and
Dr F. Mitjans (Merck Biomedical Research Laboratory, Barcelona, Spain),
respectively. MoAb 93.1B3 (anti-CD20) was kindly provided by Dr R. Vilella (Hospital Clínic, Barcelona, Spain). Anti-MMP-2
(Ab-3), polyclonal Ab anti-MMP-14 (Ab-2), anti-TIMP-1 (Ab-2), and
anti-TIMP-2 (Ab-1) were purchased from Oncogene Research Products
(Cambridge, MA).
Human plasma fibronectin was obtained from Sigma Chemical Co (St Louis,
MO). Mouse laminin-1 was a generous gift from Dr H.K. Kleinman
(National Institute of Dental Research, Bethesda, MD). Type-I and
type-IV collagen, as well as polylysine were purchased from
Collaborative Research (Bedford, MA). Fibronectin-derived synthetic
peptides GRGDSPC and EILDVPST and control peptide GRGES were obtained
from Peninsula Laboratories (Belmont CA).
Cytochalasin D, EGTA, herbymicin A, pertussis toxin, phorbol
12-myristate 13-acetate (PMA), and p-aminophenylmercuric acetate (APMA)
were purchased from Sigma Chemical Co. Wortmannin,
2'amino-3'methoxy-flavone (PD 98059), and
4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)1H-imidazole (SB202190) were obtained from Calbiochem-Novabiochem (La Jolla, CA).
Cell culture.
Human T-cell lines (CCRF-CEM, Jurkat, Molt-4) were obtained from the
European Collection of Cell Cultures (Salisbury, UK) and grown in RPMI
1640 (GIBCO Life Technologies, Grand Island, NY) supplemented with 10%
fetal calf serum (FCS), 2 mmol/L glutamine, and 50 µg/mL gentamycin
at 37°C, 5% CO2, up to a maximal cell density of 8 × 105 cells/mL at which point the cells were passed
1:10. Unless otherwise indicated, cells were used in experiments at a
concentration of 2 to 3 × 105 cells/mL. Human
umbilical vein endothelial cells (HUVEC) were obtained from freshly
delivered cords and grown as reported.28 Cells at passage 4 to 6 were used for experiments.
Adhesion assays.
Extracellular matrix proteins were diluted in phosphate-buffered saline
(PBS) at 100 µg/mL. 96-well tissue culture plates were coated with
either laminin-1, type-I collagen, type-IV collagen, fibronectin, or
polylysine at 5 µg/well. Laminin-1 was incubated for 1 hour at
37°C whereas fibronectin, type-I collagen, type-IV collagen, and
polylysine were incubated overnight at 4°C. Then, the remaining
fluid was aspirated. Cells were suspended in serum-free RPMI medium,
placed on coated plates at 1.5 × 105 cells/well and
incubated at 37°C for 90 minutes. In some experiments, cells were
preincubated in suspension with the mentioned chemicals for 30 minutes
at 37°C before the attachment step was performed. Nonadherent cells
were removed by aspiration and wells were washed once with warm,
serum-free medium. Adherent cells were fixed and stained with 0.2%
crystal violet in 20% methanol in PBS for 20 minutes and then washed
repeatedly with distilled H2O. After solubilization with
1% SDS, the optical density was measured with a spectrophotometer at
600 nm wavelength.
Gelatin zymography.
Lymphoblastoid cell lines were washed and resuspended in serum-free
RPMI medium and cultured in suspension or in dishes coated with
different extracellular matrix proteins or fibronectin-derived synthetic peptides. Alternatively, in some experiments fibronectin or
synthetic peptides were added in solution. Twenty-four hours later, the
conditioned medium was collected, centrifuged, and stored at
 20°C. In some experiments, cells were preincubated with
purified MoAb at 10 µg/mL for 30 minutes at 4°C. In additional experiments, cells were incubated at 37°C with chemicals.
Cytochalasin D was applied at 20 µmol/L; EGTA at 1 mmol/L; herbimycin
A at 2 µmol/L; pertussis toxin at 1µg/mL; PMA at 10 ng/mL;
wortmannin at 10 nmol/L; PD98059 at 5 µmol/L; and SB 202190 at 0.5 µmol/L. These products were added 30 minutes before exposure to
fibronectin. Experiments with chemicals were run for only 6 hours to
ensure cell viability that was always confirmed by trypan blue exclusion.
The conditioned medium obtained from 107 cells was
concentrated 200-fold using Urifil-10 concentrator devices (Millipore,
Molsheim, France) at 4°C, and subjected to SDS-PAGE through 10%
polyacrylamide gels copolymerized with 0.2 g/mL gelatin (Bio-Rad
Laboratories, Hercules, CA). Gels were washed with 2.5% Triton-X-100,
rinsed with 10 mmol/L Tris and incubated overnight at 37°C in 50 mmol/L Tris, 5 mmol/L CaCl2, and 1 µmol/L
ZnCl2. Gels were fixed and stained with 0.2% Coomassie
blue R250. After destaining, gelatinolytic signals were quantified by
densitometry (Gel Analysis Program SW5000 software, Ultraviolet
Products, Cambridge, UK).
Unconcentrated conditioned medium from HUVEC was used as a control in
some zymograms either freshly obtained or treated with 1 mmol/L p-APMA
for 3 hours at 37°C.
Western blot analysis.
Conditioned medium from 108 CEM cells per condition was
passed through a 10 mL gelatin-Sepharose column (Sigma). The column was
washed with 10 volumes of 50 mmol/L Tris, pH 7.6, 0.5 mol/L NaCl, 5 mmol/L CaCl2, 0.02% Brij35 (Sigma) and the gelatin-bound proteins were eluted with 5 volumes of 50 mmol/L Tris, pH 7.6, 0.5 mol/L NaCl, 5 mmol/L CaCl2, 0.02% Brij35, and 10%
dimethyl sulfoxide (DMSO). The eluted solution was dialyzed against 50 mmol/L Tris, pH 7.6, 0.2 mol/L NaCl, 5 mmol/L CaCl2 , 0.02% Brij35, and concentrated.
Concentrated samples were electrophoresed under nonreducing conditions
through a 10% polyacrylamide gel and transferred to a methanol-treated
Hybond-PVDF membrane. After an overnight blocking step with 5% nonfat
powdered milk in tris-buffered saline (TBS) with 0.05% Tween 20 (TBS-T) at 4°C, the membrane was incubated with 1 µg/mL of Ab-3
(anti-MMP-2) MoAb in 0.05% nonfat powdered milk in TBS-T for 1 hour at
room temperature. After two washes with TBS-T, the membrane was
incubated with an horseradish peroxidase (HRP)-conjugated goat
antimouse polyclonal antibody at 0.2 µg/mL for 1 hour at room
temperature, washed as above, incubated 1 minute with ECL
Western blotting detection reagants (Amersham International plc, Little
Chalfont, UK) and exposed to x-ray film.
Flow cytometry.
CEM cells (105 per condition) were incubated with either
anti-MMP-2, anti-TIMP-1, anti-TIMP-2, or anti-MMP-14 at 5 µg/mL
in PBS containing 2% FCS and 0.01% NaN3 for 20 minutes at
4°C. Cells were washed twice in 2% FCS, 0.01% NaN3 in
PBS, and incubated with a biotinylated rabbit antimouse antibody
(Dakopatts, Denmark) at 1:100 dilution for 20 minutes at
4oC. After two washes, cells were incubated as above with
Streptavidin Tricolor fluorescent conjugate (Caltag Laboratories,
Burlingame, CA) diluted 1:100 and washed. Fluorescence was quantified
with a FACStar Plus fluorescence activated cell sorter. The point 0 of
the scale was adjusted to the background fluorescence displayed by
biotinylated bovine serum albumin (BSA).
Reverse transcriptase-polymerase chain reaction (RT-PCR).
Total cellular RNA was extracted from 2 × 107 CEM
cells by lysis with 4 mol/L guanidin-isothiocyanate,
ultracentrifugation through a CsCl gradient, and phenol-chloroform
extraction.28 First-strand complementary DNA (cDNA) was
synthesized from 5 µg of RNA with T-primed first-strand kit
(Pharmacia Biotech Inc, Upsala, Sweden) according to the
manufacturer's instructions. A 1:100 final dilution of the reaction
was used as a template for PCR.
Oligonucleotide primers for PCR amplification were designed according
to published sequences for MMP-229 and MMP-1414 spanning one intron-exon junction. MMP-2 upstream primer was 5' GGC ACC CAT TTA CAC CTA CAC CAA 3' (position 1216-1239) and MMP-2 downstream primer was 5' GCT TCC AAA CTT CAC GCT CTT CAG 3'
(position 1909-1886). The predicted size of the amplification product
was 694 bp. MMP-14 upstream primer was 5' CTC CTG CTC CCC CTG CTC ACG 3' (position 142-162). MMP-14 downstream primer was 5'
CTC ACC CCC ATA AAG TTG CTG 3' (position 969-949). The expected
size of the amplicon was 828 bp. TIMP-1 and TIMP-2 were amplified using previously published primers.23,24 Amplification of
2-microglobulin cDNA was used as control of template loading among conditions.
Amplification of MMP-2, TIMP-1, TIMP-2, and 2-microglobulin cDNA was
performed with 0.5 µmol/L of each primer, 0.25 mmol/L dNTPs, 1 U of
Expand High-Fidelity PCR System (Roche Diagnostics GmbH,
Germany) enzyme mix and a 1:10 dilution of the reaction buffer supplied by the manufacturer. Amplification of MMP-14 cDNA was
performed with 0.25 mmol/L dNTPs, 0.5 µmol/L of each primer, 1 U
of Expand enzyme mix, 15 mmol/L NH4SO4,
1.5 mmol/L MgCl2, and 60 mmol/L Tris, pH 9.5. After a
hotstart step, in which the polymerase was added, each cycle consisted
of 30 seconds of denaturation at 95oC, 1 minute of
annealing at 55°C (TIMP-1, 2-microglobulin), 58°C (TIMP-2),
or 60°C (MMP-2, MMP-14), and 1 minute of extension at 72°C.
Reactions were run for 25 cycles (2-microglobulin), 35 cycles
(TIMP-1, TIMP-2), or 40 cycles (MMP-2 and MMP-14), followed by a final
elongation step of 10 minutes.
The specificity of the PCR reactions was confirmed with either nested
PCR or endonuclease restriction analysis.
Transfection of CEM T cells.
The plasmid pMMTVrasH (Asn-17) was kindly provided by Dr E. Santos
(National Cancer Institute, Bethesda, MD). pMMTVrasH (Asn-17) is an
expression plasmid in which the dominant negative mutant of Ha-Ras
Asn-17 is under control of a mouse mammary tumor virus LTR and is
inducible by dexamethasone. The plasmid contains also a
Neor gene constitutively expressed from the simian virus 40 early promoter.30 CEM cells were transfected using DMRIE-C
reagent (GIBCO Life Technologies) according to the instructions of the manufacturer. Transfected cells were exposed to fibronectin for 24 hours in serum-free conditions and the supernatant fluid was subjected
to gelatin zymography as described above. When the expression of Ha-Ras
Asn 17 was desired, transfected cells were exposed to dexamethasone
(Calbiochem-Novabiochem) at 0.5 µmol/L for 24 hours before exposure
to fibronectin, being sustained during the assay as well.
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RESULTS |
Fibronectin induces gelatinase A (MMP-2) and gelatinase B (MMP-9)
activities in lymphocyte cell line supernatants.
In serum-free conditions, CEM, and Jurkat T-cell lines transiently
adhered to various extracellular matrix proteins (laminin-1, fibronectin, type-IV collagen) and to adhesion-supporting polymers such
as polylysine. The strongest adhesion was observed on fibronectin in
which CEM and Jurkat T cells fully spread. Maximal adhesion was
observed at 60 to 90 minutes and decreased thereafter (data not shown).
Zymography analysis of the conditioned media obtained from cells plated
on fibronectin showed the presence of gelatinase activity that was
absent from the media conditioned by cells plated on other
extracellular matrix proteins (Fig 1A). Two
major bands at 92 kD and 72 kD were observed consistent with the
zymographic pattern of gelatinase B (MMP-9) and gelatinase A (MMP-2)
proenzymes, respectively.
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| Fig 1.
(A) MMP-2 and MMP-9 induction by fibronectin. CEM T cells
were cultured on several extracellular matrix proteins for 24 hours.
The figure shows the zymographic pattern of concentrated conditioned
medium of 107 cells plated on polylysine (lane 1),
fibronectin (lane 2), laminin-1 (lane 3), type-I collagen (lane 4), and
type-IV collagen (lane 5). (B) Accumulation of MMP-2 and MMP-9 in the
conditioned medium over time. 107 cells were incubated on
plastic or on fibronectin for different periods of time. The
conditioned medium was concentrated and subjected to gelatin
zymography. The figure shows the gelatinolytic signal quantified as
described in the Materials and Methods section. (C) CEM cells produce
activated forms of MMP-2 in response to fibronectin. Unconcentrated
conditioned medium obtained from HUVEC confluent monolayers cultured on
plastic was subjected to gelatin zymography in the absence () or in
the presence (+) of p-aminophenylmercuric acetate (APMA).
Concentrated conditioned medium from 107 CEM cells cultured
on fibronectin (Fn) for 24 hours disclosed an identical zymographic
pattern.
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Gelatinase B was sometimes detected in conditioned medium from CEM
cells grown on uncoated plastic. In contrast, traces of gelatinase A
could only be observed when cells were maintained for long incubation
periods (Fig 1B). Gelatinase A and B production increased dramatically
after a few hours of exposure to fibronectin and continued accumulating
in the conditioned medium over a 48-hour period (Fig 1B). Even though
the specific activity of gelatinase B is 25 times higher than the
gelatinase A one,22 the intensity of the gelatinase A
gelatinolytic signal elicited by fibronectin was severalfold over that
for gelatinase B (Fig 1A and B). In addition, the increase in
gelatinolytic signal induced by 48-hour exposure to fibronectin was
30-fold for gelatinase A and only threefold for gelatinase B (Fig 1B).
The absolute amount of gelatinase A and B released to the supernate,
even after exposure to fibronectin, was very small. Their zymographic
detection required 200 times concentration of the conditioned medium
and both MMP-2 and MMP-9 were repeatedly undetectable by enzyme-linked
immunoassay of nonconcentrated medium using high sensitivity (pg/mL)
commercial kits (Biotrak; Amersham).
Although gelatinase B (MMP-9) synthesis by T lymphocytes has been
repeatedly recognized,21-25 gelatinase A (MMP-2) production has only been identified under very restricted
conditions.22,26 To further characterize the lower
molecular weight species observed in gelatin zymograms of
T-cell-conditioned media, a comparative zymogram was performed with
conditioned media from HUVEC treated with APMA. The production of the
72 kD gelatinase-A proenzyme by HUVEC has been well
characterized31 and APMA induces the activation of MMPs by
promoting the autolytic cleavage of N-terminal peptide sequences. As
shown in Fig 1C, the low molecular weight gelatinolytic bands obtained
from CEM supernates were identical to the 62 and 59 kD products
obtained by APMA treatment of HUVEC-conditioned medium. Additional
confirmation that the 72 kD gelatinolytic band and the lower molecular
weight species corresponded to MMP-2 was obtained by Western blot
analysis. Given the lower sensitivity of Western Blot compared with
gelatin zymography, a previous gelatin-affinity purification step of
conditioned medium was required for MMP-2 detection (Fig
2A). Western blotting of
gelatin-affinity-purified HUVEC-conditioned medium, used as control,
displayed only the 72 kD proenzyme (data not shown).
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| Fig 2.
(A) Western blot identification of MMP-2 in
CEM-conditioned medium. Gelatin affinity-purified-conditioned medium
from 108 CEM cells cultured for 24 hours on plastic (P) or
in the presence of soluble fibronectin at 10 µg/mL (Fn) was subjected
to Western blot analysis with the MoAb Ab-3 against MMP-2. (B) RT-PCR
demonstration of MMP-2 transcripts in CEM cells. PCR-amplified cDNA
obtained from resting CEM cells (lane 1) and CEM cells exposed to
fibronectin at 10 µg/mL for 4 hours (lane 2). Simultaneous
amplification of the housekeeping gene 2-microglobulin is shown to
ensure an equivalent amount of template in both conditions. Lane 3 shows the nested PCR product obtained from the 694 bp fragment using
previously published internal primers.22 Lane 4 shows the
Sac I restriction fragments of the 694 bp fragment.
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To determine whether fibronectin induced MMP-2 transcription or just
its release into the supernatant fluid, the presence of MMP-2 messenger
RNA (mRNA) was investigated by RT-PCR. As shown in Fig 2B, MMP-2 mRNA
was barely detectable in cells in suspension, whereas a clear band of
the expected size was detected in cells exposed to fibronectin. Nested
PCR and restriction fragment analysis confirmed the specificity of the
PCR amplification of MMP-2.
Fibronectin induction of MMP-2 is mediated by interaction with
4, 5, and v-bearing
lymphocyte integrins.
To investigate which fibronectin receptors participate in
fibronectin-mediated induction of MMP synthesis, CEM T cells were preincubated with blocking MoAb against several integrins. As shown in
Fig 3A, blocking 4,
5, and v but not 6 chains
dramatically reduced MMP-2 production. A less-apparent reduction in
MMP-9 was also observed. Anti-6 integrin chain (a
laminin receptor) and anti-CD20 (a B-cell lineage marker) were used as
negative controls.
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| Fig 3.
(A) Inhibition of fibronectin-induced MMP-2 and MMP-9 by
blocking MoAb against integrin chains. 107 CEM cells
per condition were preincubated with medium alone or with MoAb against
4, 5, and V integrin
chains (fibronectin receptors); 6 integrin chain (a
laminin receptor); or the anti-CD20 93.1B3 (a B-cell lineage marker)
before exposure to fibronectin. Conditioned medium was concentrated and
subjected to gelatin zymography. Graph shows variation in gelatinolytic
signals compared with the signal obtained with untreated cells exposed
to fibronectin. (B) Membrane-associated MMP-2 expression induced by
fibronectin-derived synthetic peptides. CEM cells were exposed to
GRGDSPC, EILDVSPT, a combination of both, or to the control peptide
GRGES at 100 µg/mL for 16 hours. Histograms show distribution of
fluorescence intensity in 5 × 103 cells per condition
incubated with the MoAb Ab-3 recognizing MMP-2. (C) Pro-MMP-2 and
pro-MMP-9 production stimulated by fibronectin-derived synthetic
peptides. 2 × 107 CEM cells were cultured on plastic
(lane 1) or on plastic coated with GRGDSPC (lane 2), EILDVSPT(lane 3),
or both (lane 4) at 100 µg/mL. The conditioned medium obtained after
48 hours was concentrated and subjected to gelatin zymography.
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To further evaluate the role of lymphocyte integrins in MMP production,
T cells were incubated with synthetic peptides containing minimal
fibronectin sequences recognized by v and
5 (RGD) and 4 (LDV) integrins. As shown
in Fig 3B, GRGDSPC and EILDVPST fibronectin peptides but not the
control peptide GRGES increased the expression of membrane-associated
MMP-2, as assessed by flow cytometry. However, when added alone,
synthetic peptides were not able to elicit the strong MMP-2 and MMP-9
secretion achieved by fibronectin. Only at saturating
concentrations32 and with long incubation periods MMP-9 and
MMP-2 proenzymes could be detected as 92 kD and 72 kD gelatinolytic
bands, respectively, and no activated forms were observed (Fig 3C).
These findings indicate that ligand occupancy is not sufficient to
elicit full production, secretion, and activation of MMPs and that
ligand clustering or concomitant interaction with other fibronectin
sequences are required.32
Fibronectin does not influence TIMP-1 production but downregulates
TIMP-2 expression by T-cell lines.
Given the importance of TIMPs on the biological activity of MMPs, we
studied whether TIMP-1 and TIMP-2 were influenced by fibronectin. In
resting CEM cells, we found constitutive mRNA expression of both TIMP-1
and TIMP-2 (Fig 4A). When CEM cells were
exposed to fibronectin a decrease in the mRNA level of TIMP-2 could be
observed, whereas the amount of TIMP-1 mRNA remained unaltered (Fig
4A). We further investigated the effect of fibronectin-derived peptides
on membrane-associated TIMP-1 and TIMP-2 by flow cytometry analysis
(Fig 4B). A baseline membrane expression of both TIMPs was found in CEM
incubated with the control peptide GRGES, being the level of TIMP-1
relatively higher than that of TIMP-2. Neither GRGDSPC nor EILDVPST
were able to modify the expression of any TIMP. However, their
combination induced a clear downregulatory effect on the surface
expression of TIMP-2, whereas no effect on TIMP-1 membrane expression
was observed.
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| Fig 4.
(A) RT-PCR analysis of TIMP-1 and TIMP-2 mRNA levels in
CEM cells stimulated with fibronectin. cDNA obtained from resting CEM
cells (lane 1) or stimulated with soluble fibronectin at 10 µg/mL for
4 hours (lane 2). 2-microglobulin amplification is used to control
template quantity in each condition. (B) Membrane expression of TIMP-1
and TIMP-2 in CEM cells treated with fibronectin-derived peptides. Flow
cytometry analysis of CEM cells cultured with a combination of GRGDSPC
and EILDVSPT in solution at 100 µg/mL for 16 hours. The peptide GRGES
was used as control. Histograms show distribution of fluorescence
intensity in 5 × 103 cells per condition.
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Fibronectin induces the expression of MT1-MMP (MMP-14) by T-cell
lines.
Because CEM cells exposed to fibronectin secreted activated forms of
MMP-2, we investigated whether CEM cells expressed MMP-14, a
membrane-bound MMP which is believed to be one of the major activators
of MMP-214,18 and which has not been previously shown to be
expressed in lymphoid cells. MMP-14 was not expressed constitutively by
CEM cells (Fig 5A and 5B). When cells were
exposed to fibronectin, MMP-14 mRNA could be detected by RT-PCR (Fig
5A). MMP-14 surface expression was absent from resting CEM cells but could be detected by flow-cytometry when cells were treated with fibronectin-derived synthetic peptides (Fig 5B). The observation that,
even at saturating concentrations, synthetic peptides were not able to
elicit the secretion of activated forms of MMP-2, indicates that
besides MMP-14 expression, other factors may be required for full MMP-2
activation, as shown in other cell types.33
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| Fig 5.
(A) Induction of MMP-14 expression in CEM cells by
fibronectin. MMP-14 (MT1-MMP) transcripts obtained from baseline CEM
cells (lane 1) and CEM cells exposed to fibronectin (10 µg/mL) for 4 hours (lane 2). Concomitant amplification of 2-microglobulin shows
equivalent amounts of template in both samples. Lane 3 shows the Nco I
restriction fragments of the 828 bp PCR product. (B) Surface expression
of MMP-14 induced by fibronectin-derived peptides in CEM cells. CEM
cells were incubated with either GRGDSPC, EILDVSPT, a combination of
both, or the control peptide GRGES at 100 µg/mL for 16 hours and
subjected to flow cytometry. Histograms show fluorescence intensity
distribution among 5 × 103 cells immunostained with the
polyclonal antibody Ab-2 recognizing MMP-14.
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|
Fibronectin-activated signaling pathways provide both stimulatory and
inhibitory signals for MMP-2 and MMP-9 expression.
As stated above, in serum-free conditions, CEM cell adhesion and
spreading on fibronectin was transient. Disrupting Rho-dependent actin
cytoskeleton organization triggered by attachment to fibronectin by
cytochalasin D34 increased both MMP-2 and MMP-9 production (Fig 6A). Moreover, when cells were exposed
to soluble fibronectin in suspension, a much higher production of both
MMP-2 and MMP-9 was detected (Fig 6B). However, although accelerated
detachment induced by EGTA strongly promoted MMP-2 production, MMP-9
production was inhibited, indicating that fibronectin-induced signaling
pathways leading to MMP-2 and MMP-9 expression are partially
independent (Fig 6A). According to this concept, protein kinase C (PKC)
activation by PMA was not able to increase fibronectin-induced MMP-2
production whereas fibronectin-induced MMP-9 expression was strongly
increased by PMA (Fig 6A). PMA alone was also able to elicit MMP-9
production by CEM cells without exposure to fibronectin, as it has been
previously described in other lymphoid cell lines.21
However, treatment with calphostin C did not result in an inhibition of
fibronectin-induced MMP-9 (Fig 6A), indicating that induction of MMP-9
by fibronectin does not occur through PKC-dependent signaling pathways.
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| Fig 6.
(A) Changes in fibronectin-induced MMP-2 and MMP-9
production achieved by manipulating integrin-mediated signal
transduction pathways. 107 CEM cells were exposed to the
above-described chemicals 30 minutes before exposure to fibronectin.
Conditioned medium was obtained after 6 hours, concentrated, and
subjected to gelatin zymography. Graph shows the variation in the
intensity of the fibronectin-induced gelatinolytic signal induced by
treatment with the different products and their simultaneous effect on
cell adhesion, both expressed as a percentage of those obtained by
fibronectin alone (labeled basal). (B) Intensity of MMP-2 and MMP-9
production induced by exposure to soluble or solid-phase fibronectin.
107 CEM cells were exposed to soluble (10 µg/mL) or
solid-phase (plastic surfaces coated with 100 µg/mL) fibronectin for
24 hours. Conditioned media were concentrated and subjected to gelatin
zymography. (C) Fibronectin-induced MMP-2 and MMP-9 production by cells
transfected with a dominant negative mutant of Ha-Ras. 107
CEM cells transfected with Ha-Ras-Asn 17, cloned in the
dexamethasone-inducible vector pMMTV, were exposed to soluble
fibronectin for 24 hours. Conditioned medium from transfected cells was
concentrated and subjected to gelatin zymography. Lane 1 shows absence
of gelatinolytic activity in conditioned medium from transfected cells
stimulated with 0.5 µmol/L dexamethasone without exposure to
fibronectin. Lanes 2 and 3 display gelatinolytic signals provided by
conditioned medium from untreated transfected cells (lane 2) and from
transfected cells treated with 0.5 µmol/L dexamethasone (lane 3),
both exposed to fibronectin. (D) Effect of PI-3K, MEK-1/2, and p38 MAPK
inhibition on fibronectin-induced MMP-2 and MMP-9 production. 2 × 107 CEM cells were preincubated with several very specific
inhibitors of PI-3K (wortmannin), ERK-1/2 kinase (PD 98059), or p38
MAPK (SB 202190) for 30 minutes before exposure to soluble fibronectin
at 10 µg/mL. Conditioned medium was obtained 6-hours later,
concentrated, and subjected to gelatin zymography. The picture shows
the gelatinolytic signals provided by conditioned medium from untreated
cells exposed to fibronectin (lane 1), or pretreated with PD 98059 (lane 2), wortmannin (lane 3), or SB 202190 (lane 4).
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Adhesion of different cell types to fibronectin activates the
Ras/Raf-1/Mitogen-activated protein kinase (MAPK), Phosphatidil inositol-3 kinase (PI-3K), and p38 MAPK signaling pathways through integrin-mediated signal transduction.35-37 CEM
transfection with the dominant inhibitory mutant Ha-Ras Asn-17
increased fibronectin-induced MMP-2 and MMP-9 production (Fig 6C).
Because transfected cells were exposed to dexamethasone to induce
Ha-Ras Asn-17 expression, the effect of dexamethasone on fibronectin
induction of the MMPs was tested. Dexamethasone alone had no effect on
fibronectin-induced MMP-2 or MMP-9 expression (data not shown). Ha-Ras
Asn-17 expression did not stimulate MMP-2 or MMP-9 production without
exposure to fibronectin (Fig 6C). Inhibition of PI-3K by wortmannin,
MAPK kinase 1 and 2 (MEK-1/2) by PD98059, and p38 MAPK by SB 202190 all
strongly increased fibronectin-induced MMP-2 and MMP-9 production (Fig
6D), indicating that signals transduced through PI-3K and MAPKs inhibit
MMP-2 and MMP-9 production by T lymphocytes. None of these inhibitors
by themselves were able to induce MMP-2 or MMP-9 without exposure to
fibronectin (data not shown). By contrast, herbimycin A, an inhibitor
of Src-type tyrosine kinases, strongly inhibited both MMP-2 and MMP-9
production (Fig 6A). None of the kinase inhibitors substantially
modified cell attachment, indicating that they influence
integrin-signaling pathways involved in MMPs production that are
independent or are located downstream of the signaling pathways
involved in cell attachment and spreading.
The existence of PI-3K- and MAPK-transduced inhibitory pathways
suppressing MMP-2 and MMP-9 production was further supported by the
observation that cells cultured at high density (1 × 106/mL or more) lost their ability to produce MMP-2 and
MMP-9 when exposed to fibronectin (Fig 7A).
Inhibition of MMP-2 and MMP-9 production by overgrown cells was not
produced by the accumulation of a soluble inhibitory factor because
conditioned medium obtained from cells cultured at high density did not
downregulate MMP-2 or MMP-9 production by cells plated on fibronectin
at low density (data not shown). Moreover, the lack of response to
fibronectin was not due to a downregulation of 4,
5, or v integrin expression because their
surface expression in cells at high density was identical to that
observed in cells plated at low density, as assessed by flow cytometry
(Fig 7B). Similarly, it was not caused by a decrease in integrin
avidity because adhesion and spreading of cells on fibronectin was even
higher in cells cultured at high density compared with that of cells
plated at low density (Fig 7A). Taken together, these data indicate a
dominance of pathways inhibiting MMP-2 and MMP-9 expression in
overgrown cells.
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| Fig 7.
(A) Abrogation of MMP-2 and MMP-9 production in response
to fibronectin by high-cell density and its reversion by inhibiting
PI-3K and different elements of the Ras/MAPK-signaling pathway. 2 × 107 CEM cells were cultured at a 2 × 105
cells/mL or at 1 × 106 cells/mL and exposed to
fibronectin. Conditioned medium was obtained after 6 hours,
concentrated, and subjected to gelatin zymography. Graph shows the
intensity of the gelatinolytic band in untreated cells (baseline),
cells expressing Ha-Ras Asn-17, and cells treated with wortmannin, PD
98059, or SB 202190. Concomitant effect on cell binding to fibronectin
is also shown. Both adhesion and intensity of the gelatinolytic bands
are expressed as fold increase or decrease over that observed with the
untreated cells exposed to fibronectin at low density. (B) Effect of
cell density on integrin cell-surface expression. Flow cytometry
analysis of membrane expression of fibronectin receptors in CEM cells
cultured at low density (filled histograms) versus cells cultured at
high density (clear histograms). Histograms show fluorescence-intensity
distribution among 5 × 103 cells immunostained with MoAb
raised against several integrin chains and the common chain.
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CEM transfection with a dominant inhibitory mutant of Ha-Ras,
inhibition of PI-3K by wortmannin, of MAPKK by PD98059, and of p38 MAPK
by SB 202190, all restored the ability of overgrown cells to produce
MMP-2 and MMP-9 in response to fibronectin (Fig 7A). Together, these
findings support the concept that integrin-mediated signaling pathways
transduce both activating and repressing signals for MMP-2 and MMP-9
production and their relative predominance is controlled by additional
factors, probably related to cell migration and cell growth.
| |
DISCUSSION |
Tissue infiltration by lymphocytes requires transmigration through
endothelial cell junctions and, subsequently, through the basement
membrane and interstitial matrix.1,7 This later step likely
requires adhesion to extracellular matrix proteins and their focalized
degradation.7,9,10 In the present study, we show that
interaction with fibronectin induces the synthesis, activation, and
secretion of MMP-2 and MMP-9 by lymphoid cell lines from T lineage. In
addition, fibronectin downregulates TIMP-2 expression, a process that
may enhance the proteolytic activity of MMP-2.
Previous studies have shown the ability of extracellular matrix
proteins to induce their own degradation by promoting the production
and release of several matrix metalloproteinases by several adherent
cell types.38-43 In our study, intact fibronectin induced
MMP-2 and MMP-9 expression whereas fibronectin-derived peptides
containing an RGD or an LDV sequence elicited only a slight response,
even at saturating concentrations, indicating that receptor occupancy
alone is not able to optimally induce MMP-2 and MMP-9 and/or that other
sequences in fibronectin are also required for optimal MMP-2 and MMP-9
production by T cells.
Fibronectin induced the secretion of activated forms of MMP-2 by
lymphoid cell lines that often predominated over the 72 kD proenzyme
form. Previous reports showing MMP-2 production by T lymphocytes
triggered by soluble factors such as IL-2 have only provided
zymographic detection of the 72 kD proenzyme with no evidence of
activated forms.23 By contrast, the 62 kD activated form
has been detected in T lymphocytes adherent to VCAM-1 expressing endothelial cells,26 a process probably mediated also by
4 integrins. Recently, the membrane-associated
metalloproteinase MT1-MMP (MMP-14) has been shown to be one of the
major activators of MMP-2 in invasive tumors14,19 and their
coordinated expression has been shown in different settings such as
development,44 angiogenesis,42 and tumor
progression.14,45 Several soluble factors such as
concanavalin A and phorbol esters can upregulate MMP-14
production.33,46 Herein, we show for the first time that
T-cell lines, indeed, produce MMP-14 and that, in addition to soluble
factors, interactions with matrix molecules such as fibronectin are
able to induce MMP-14 expression.
In our study, fibronectin-induced MMP-2 and MMP-9 production by T-cell
lines was mediated by signaling triggered by engagement of
4, 5, and v-bearing
integrins that have all been shown to serve as fibronectin
receptors.47 Integrins mediating MMP induction by matrix
proteins, as well as the type of MMP produced vary among different cell
types.41,43,48 In synovial fibroblasts, for instance,
51 engagement induces MMP-1 and MMP-3
expression whereas 41 seems to have an
inhibitory role on 51-mediated MMP
expression.38,43 A predominant function of
41 in this cell type might then account for the inability of intact fibronectin to induce MMP expression by
synovial fibroblasts.43 41
has virtually no role in fibronectin-induced MMP-9 expression by
macrophages.49 By contrast, according to our results,
41 seems to induce MMP-2 and MMP-9
expression by T lymphocytes because blocking MoAb against
4 highly reduced the MMP production induced by intact
fibronectin. Recent work has shown the production of MMP-2 by
lymphocytes adherent to endothelial cells expressing VCAM-1 and to
recombinant VCAM-1 itself,26 further supporting the concept
that engagement of 41 triggers MMP-2
production by T lymphocytes.
Integrins mediate a variety of important cell functions such as
adhesion, migration, anchorage-dependent growth, and gene expression.47 The complex signaling pathways triggered by
integrin interactions with their ligands are beginning to be
understood. Integrins activate small-G proteins of the Rho subfamily
that regulate focal adhesion formation and actin cytoskeleton
organization.50 Rho, in turn, activates, PI-5K and the MAPK
JNK, whereas other members of the Rho subfamily, Rac-1 and cdc42,
activate PI-3K.51 Inhibition of small-G protein activation
by cytochalasin D34 led to an increase in
fibronectin-induced MMP-2 and MMP-9 indicating that small-G proteins of
the Rho subfamily transduce inhibitory signals for gelatinase
induction. Integrin-mediated cell adhesion leads to PKC
activation.52 However, PKC is not probably involved in
MMP-2 expression because phorbol esters did not modify MMP-2 production
by T lymphocytes in accordance with the absence of phorbol-responsive
elements in the MMP-2 gene.53 PKC activation by phorbol
esters is known to induce MMP-9 expression in a variety of cell types,
including T lymphocytes and phorbol-responsive elements, including AP-1
binding sites, have been well characterized in the MMP-9
promoter.54 Nevertheless, PKC activation is not probably
involved in fibronectin-induced MMP-9 expression by T-cell lines,
because PKC inhibition by calphostin C did not result in a reduction of
fibronectin-induced MMP-9 production. According to this observation it
has been shown that T-lymphocyte attachment to fibronectin activates
AP-1 in a PKC-independent manner.55
Integrin activation results in phosphorylation of the module protein
Shc creating binding sites for the adaptor protein Grb-2, which
recruits the guanidine nucleotide exchanging factor Sos activating the
Ras/Raf-1/MAPK (Erk-1 and -2)-signaling cascade.35 Integrin engagement may also activate Erks through PI-3K activation downstream of Ras.37 Furthermore, activation of p38 MAPK
can also be triggered by integrin-mediated signaling through
independent pathways.35 In our system, inhibition of Ha-Ras
by transfection with a dominant negative mutant, and inhibition of
PI-3K and MAPKs increased fibronectin-induced MMP-2 and MMP-9
expression suggesting that inhibitory signals for the expression of
these MMPs are transduced through Ras/Raf-1/MAPK pathways. Upstream
inhibition of the MAPK cascade by the G-protein inhibitor pertussis
toxin also resulted in an increased MMP-2 and MMP-9 production.
Finally, integrin-mediated signaling leads to tyrosine phosphorylation
of FAK which, in turn, creates sites for interaction with proteins
displaying SH2 domains such as Src-type tyrosine kinases.56
Inhibition of Src-type tyrosine kinases with herbimycin A significantly
reduced MMP-2 and MMP-9 expression without a significant change in cell
adhesion suggesting an important role for Src-type tyrosine kinases in
integrin-mediated signaling leading to MMP-2 and MMP-9 expression by T lymphocytes.
Gelatinase expression is regulated by cytokines, growth factors, and
matrix components through complex signaling pathways that are beginning
to be understood. Mechanisms involving fibronectin-induced gelatinase
expression by T-lymphoid cell lines largely differ from those mediating
gelatinase production by transformed cells derived from solid tumors.
Some transformed cell lines constitutively express MMP-9, MMP-2, or
both54,57,58 and activated Ras seems to play a role in
spontaneous gelatinase expression by some of them.56,59 It
has been shown that v-Src can activate the expression of MMP-9 through
mechanisms independent from that triggered by some cytokines and growth
factors.60 According to our data, fibronectin induction of
gelatinases in T-cell lines was mediated through integrin-mediated
signaling pathways probably involving Src-type tyrosine kinases and not
by other pathways employed by G-protein-coupled growth-factor
receptors, which, in fact, had an inhibitory function. Contrarily to
other cell responses,36 T-lymphocyte integrins do not
cooperate with growth factors in gelatinase induction.
When undisrupted, cell interaction with fibronectin promotes cell
attachment and spreading. As mentioned, integrin-mediated adhesion to
fibronectin leads to the activation of the MAPK cascade in different
cell types.35-37 MAPK activation, in turn, has been shown
to be crucial for cell migration through complex and largely unknown
mechanisms, among which direct phosphorylation of myosin light chains
has been recently identified.61 On the other hand, excessive adherence to substrates reduces cell motility, and
circumstances that halt integrins in an adherent status lead to a
decreased cell migration.59 Recently, it has been shown
that MAPKs mediate an interesting self-inhibitory loop. Excessive MAPK
activation achieved by transfecting cells with constitutively active
H-Ras or Raf-1 decreases integrin avidity.59 Decreasing
integrin avidity by activated MAPKs may allow the coordinated series of
adhesion-deadhesion events required for cell motility. In our system,
lymphocyte adhesion to fibronectin was transient suggesting the
existence of a similar kind of negative feedback triggered by
interaction with fibronectin itself. Similarly, in our setting,
adhesion to fibronectin was a necessary stimulus for MMP-2 and MMP-9
production (presumably mediated by Src-type tyrosine kinases) but
signals transduced through other pathways (PI-3K and MAPKs activation)
provided inhibitory signals for MMP-2 and MMP-9 expression. As
mentioned, several stimuli promoting cell release increased
fibronectin-induced MMP-2 and MMP-9 production by T lymphocytes whereas
a high-density status that was associated with a higher cell adherence,
suppressed MMP-2 and MMP-9 induction, suggesting a connection between
cell migration, a phenomenon requiring coordinated series of adhesion
and deadhesion events, and gelatinase production. Our findings suggest
that migrating T lymphocytes in contact with fibronectin release
tightly regulated pulses of very small amounts of activated gelatinases
which results in focalized matrix degradation, allowing their
progression through the b |
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ABSTRACT
INTRODUCTION