Center for Cell Biology and Cancer Research, Albany Medical College, Albany, New York 12208, USA
* Author for correspondence (e-mail: higginp{at}mail.amc.edu )
Accepted 14 May 2002
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Summary |
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Key words: PAI-1 transcription, Cytoskeleton, Signal transduction, Cell shape
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Introduction |
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Perturbation of cell morphology with cytoskeleton-targeting drugs provides
one important approach to the identification of shape-responsive genes and, in
some cases, the involved signaling pathways
(Aggeler et al., 1984;
Zambetti et al., 1991
;
Higgins et al., 1992
;
Bershadsky et al., 1996
;
Irigoyen et al., 1997
;
Varedi et al., 1997
;
Schmid-Alliana, 1998; Wang et al.,
1998
). The transcription of genes that encode proteins involved in
tissue remodeling processes, such as urokinase plasminogen activator (uPA) and
its type-1 inhibitor (PAI-1), is particularly closely associated with dynamic
changes in cellular morphology and shape-altering physiologic processes
(Higgins et al., 1992
;
Ryan and Higgins, 1993
;
Bayraktutan and Jones, 1995
;
Ailenberg and Silverman, 1996
;
Irigoyen et al., 1997
;
Providence et al., 2000
;
Yan et al., 2000
).
Growth-factor-initiated epithelial-to-mesenchymal transition or disruption of
E-cadherin-dependent cell-to-cell contacts and cadherin-associated actin
structures, for example, stimulates PAI-1 expression and uPA secretion
(Frixen and Nagamine, 1993
;
Zavadil et al., 2001
).
Cellular migration, both over planar surfaces and through complex `tissue'
barriers, moreover, also involves extensive morphological restructuring and is
similarly accompanied by induced PAI-1 and uPA transcription
(Pepper et al., 1987
;
Pepper et al., 1992
;
Lauffenburger and Horwitz,
1996
; Friedl and Brocker,
2000
; Providence et al.,
2000
; Ridley,
2001
). Such findings consistently link expression controls on this
protease-protease inhibitor pair to specific cytoarchitectural changes,
probably as part of the motile program
(Providence et al., 2000
), and
suggest involvement of the cytoskeleton in the signaling apparatus. Indeed,
targeted reorganization of cell morphology with the cytoskeleton-active agent
cytochalasin D (CD) does, in fact, transcriptionally activate both the uPA and
PAI-1 genes (Higgins et al.,
1992
; Lee et al.,
1993
). Microfilament-disrupting agents similarly increase
transforming growth factor-ß1 (TGF-ß1), c-fos, collagenase
and fibronectin transcription (Zambetti et
al., 1991
; Varedi et al.,
1997
), suggesting a specific genetic response to cytoskeletal
remodeling. Cell-shape-related induction of TGF-ß is particularly
relevant since PAI-1 transcription in CD-stimulated cells is a secondary
(i.e., protein-synthesis-dependent) event
(Higgins et al., 1995
), and
PAI-1 is a major TGF-ß1-inducible gene
(Boehm et al., 1999
). It was
important, therefore, to assess the potential involvement of an autocrine
TGF-ß1 loop in shape-initiated PAI-1 expression. Drug-initiated
alterations in both the microfilament and microtubule networks, moreover, also
mobilize intracellular signaling elements activating the ERK, JNK and p38
mitogen-activated protein kinases (MAPKs)
(Irigoyen et al., 1997
;
Rijken et al., 1998
;
Schmid-Alliana, 1998; Sotiropoulos et al.,
1999
; Ren et al.,
1999
; Irigoyen and Nagamine,
1999
; Yujiri et al.,
1999
; Subbaramhiah et al., 2000). The relationship between kinase
stimulation in response to cytoskeletal disruption and the associated
reprogramming of gene expression, however, remains to be defined. The present
study was designed, therefore, to identify signaling intermediates involved in
this unique pathway of PAI-1 gene regulation. Strategies were utilized to
manipulate both the actin- and tubulin-based cytoskeletons in order to
distinguish potential network-specific controls on intracellular signaling
cascades/PAI-1 expression from cell-shape-dependent events.
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Materials and Methods |
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Northern blotting
Total cellular RNA was isolated and resuspended in TE buffer; 10-15 µg
RNA was separated in 1.2% agarose/formaldehyde gels, transferred to Nytran
membranes and immobilized by UV crosslinking. Blots were incubated at 42°C
for 2 hours in 50% formamide, 5x Denhardt's reagent, 1% SDS, 200
µg/ml heat-denatured salmon sperm DNA (ssDNA), 5x SSC then hybridized
overnight at 42°C in 50% formamide, 1% SDS, 2.5% SSC, 5x Denhardt's
reagent, 100 µg/ml ssDNA, 20% dextran sulfate to 32P-labeled
PAI-1 and A50 cDNA probes. A50 was selected as a normalizing transcript since
expression of A50 is unaffected by serum, growth factors or cell-shape
perturbation (Ryan et al.,
1996; Providence et al.,
1999
; Kutz et al.,
2001
). Membranes were washed at 55°C in 0.1xSSC/0.1%SDS
and hybridization signals quantified with a Storm phosphorimager (Molecular
Dynamics, CA).
MAP kinase assay
Cells were extracted for 30 minutes in ice-cold lysis buffer (0.5%
deoxycholate, 50 mM HEPES [pH 7.5], 1% Triton X-100, 1% NP-40, 150 mM NaCl, 50
mM NaF, 1 mM vanadate, 0.01% aprotinin, 4 µg/ul pepstatin A, 10 µg/ul
leupeptin, 1mM phenylmethanesulfonyl fluoride; 1 ml/100 mm dish) and lysates
clarified at 14,000 g for 15 minutes. Extract protein
concentration was determined with the BCA Protein Assay Kit (Pierce, Rockford,
IL), and 500 µg aliquots from control and CD-treated cells were incubated
with ERK1 and/or ERK2 antibodies (2 µg each; Santa Cruz Biotechnology,
Santa Cruz, CA) for 2 hours (or overnight) in a reaction volume of 500 µl
at 4°C while rocking. Protein A/G Plus-agarose (30 µl) was added and
immune complexes collected 2 hours later by centrifugation, washed twice with
lysis buffer and twice with 100 mM NaCl in 50 mM HEPES buffer (pH 8.0).
Complexes were incubated at 37°C for 15 minutes in kinase reaction buffer
(10 µCi 32P-ATP, 50 µM ATP, 20 mM HEPES [pH 8.0], 10 mM
MgCl2, 1mM DTT, 1mM benzamidine, 0.3 mg/ml myelin basic protein
[MBP]), diluted in sample buffer (50 mM Tris-HCl, pH 6.8, 10% glycerol, 1%
SDS, 1% 2-mercaptoethanol), boiled and 15 µl aliquots separated on SDS/15%
acrylamide slab gels. Proteins were transferred to nitrocellulose, and
radiolabeled MBP was quantified by phosphorimager analysis and total MBP
assessed by Ponceau S staining. Nitrocellulose membranes were blocked in 3%
milk, incubated overnight with ERK1 and ERK2 antibodies (0.2 µg/ml),
washed, incubated with horseradish-peroxidase-conjugated secondary antibodies
(0.13 µg/ml), and the proteins were visualized by enhanced
chemiluminescence.
pp60c-src kinase assay
Cells were extracted for 30 minutes in ice-cold lysis buffer as used for
MAP kinase activity assessments (above) but containing 0.1% SDS and lysates
clarified at 14,000 g for 15 minutes. Protein aliquots (300
µg) from control and CD-treated cells were incubated with monoclonal
antibodies to pp60c-src (clone GD11; Upstate
Biotechnology, Lake Placid, NY) (2 µg) for 2 hours in a reaction volume of
300 µl. Immune complexes were collected on Protein A/G Plus-agarose (as
above) and washed twice with lysis bufer without SDS and twice with wash
buffer (20 mM HEPES pH 7.4, 10 mM MgCl2, 10 mM MnCl2, 150 mM NaCl).
Complexes were incubated at 30°C for 20 minutes in SRC kinase reaction
buffer (10 µCi 32P-ATP, 5 µM ATP, 20 mM HEPES [pH 7.4], 10 mM
MgCl2, 10 mM MnCl2, 150 mM Nacl) followed by addition of 10 µl
electrophoresis sample buffer. After boiling, 15 µl aliquots were separated
on SDS/15% acrylamide slab gels, and proteins were transferred to
nitrocellulose. Radiolabeled pp60c-src and IgG heavy chain
(a substrate for immunoprecipitated SRC kinase) were quantified by
phosphoimager analysis. pp60c-src and IgG heavy chain
levels in each lane were assessed by western blotting. Briefly, nitrocellulose
membranes were blocked in 3% milk, incubated overnight with
pp60c-src antibody (GD11); the immunoblots were then
washed, incubated with the appropriate secondary antibodies and proteins
visualized by enhanced chemiluminescence.
Microscopy
Quiescent cells were treated with CD or colchicine (for the times and at
the concentrations indicated) or DMSO and fixed in 10% formalin for
phase-contrast microscopy. For each culture condition, 50 random cells were
selected for area measurements using Image Pro-Plus analytical software;
cellular perimeters were outlined, the spread cell area (`footprint')
calculated (mean±standard deviation) and plotted as a function of
treatment. Cytoskeletal structures and intracellular PAI-1 protein were
visualized by two-color fluorescence microscopy. Formalin (10%)-fixed cells
were permeabilized in 1% NP-40/PBS for 20 minutes, incubated in 1% BSA for 20
minutes then with rabbit anti-rat PAI-1 IgG (10 µg/ml) for 1 hour and
washed three times. Cells were incubated simultaneously with Alexa-488-labeled
goat anti-rabbit IgG and rhodamine-conjugated phalloidin (1 µg/ml) to
visualize PAI-1 and microfilament organization, respectively. Coverslips were
mounted with anti-fade reagent (Molecular Probes, Eugene, OR). For analysis of
cellular microtubules, formalin-fixed cells were washed three times with PBS
(without CA+2/Mg+2) then permeabilized in 0.5% Triton
X-100/PBS for 10 minutes followed by three PBS washes. After a BSA block (as
above), cells were incubated with a monoclonal antibody to ß-tubulin (1-2
µg/ml) and rabbit anti-PAI-1 (10 µg/ml) for 1 hour at room temperature,
washed three times and incubated in Alexa-568-labeled goat anti-mouse IgG and
Alexa-488-labeled goat anti-rabbit IgG for 45 minutes to visualize tubulin and
PAI-1, respectively. Cells were rinsed for 15 minutes in PBS followed by two
additional washes for 5 minutes each, incubated in DAPI (to stain the nuclei),
washed twice and mounted with anti-fade reagent.
PAI-1 western blotting
Cells were extracted for 30 minutes in ice-cold lysis buffer (as used in
MAP kinase assays but containing 0.1% SDS) and lysates clarified at 14,000
g for 15 minutes. PAI-1 protein was detected on nylon membrane
transfers of electrophoretic separations of 20 µg of total cellular lysate
(quantified with the BCA Protein Assay Kit) by chemiluminescence using the IgG
fraction of rabbit anti-rat PAI-1 (3-5 µg/ml) and
horseradish-peroxidase-conjugated secondary antibodies (0.13 µg/ml).
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Results |
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Metabolic requirements for induced PAI-1 expression
Assessments of signaling pathways involved in PAI-1 induction were
standardized to a 4 hour exposure to 10 µM CD or colchicine
(Fig. 1). R22 cells were
incubated with metabolic inhibitors for 30 minutes prior to treatment.
CD-stimulated PAI-1 expression was actinomycin D sensitive and largely
inhibited by puromycin (Fig.
5). The increase in PAI-1 transcripts in CD-treated cultures was a
secondary (i.e., protein-synthesis-dependent) response differing from the
primary mode of PAI-1 gene activation by serum growth factors
(Ryan et al., 1996;
Boehm et al., 1999
). Neither
puromycin nor actinomycin D when used alone, moreover, affected PAI-1 mRNA
levels relative to quiescent controls (Fig.
5), supporting their suitability to assess the mechanism (i.e.,
primary versus secondary) of CD-dependent PAI-1 induction. PAI-1 expression in
response to colchicine treatment was similarly transcriptionally dependent and
sensitive to puromycin (data not shown).
|
Cytochalasin also induces TGF-ß1 transcription
(Varedi et al., 1997), and
TGF-ß1 is a potent stimulator of PAI-1 synthesis (e.g.
Boehm et al., 1999
). Since
PAI-1 expression in CD-treated R22 cells has a significant secondary component
(Fig. 5), it was important to
evaluate the potential contribution of an autocrine TGF-ß1 loop to the
inductive process. Addition of broad-spectrum TGF-ß-neutralizing
antibodies effectively blocked PAI-1 induction by exogenously added
TGF-ß1 but failed to inhibit CD-stimulated PAI-1 expression
(Fig. 5). PAI-1 transcript
abundance in CD-treated cultures, moreover, was consistently lower than the
level of PAI-1 expression evident in 1 ng/ml TGF-ß1-stimulated cells.
Thus, the range of neutralizing antibodies used (2-20 mg/ml) is probably more
than sufficient to block any PAI-1 induction that may have been mediated by an
autocrine TGF-ß-dependent pathway.
Cell-shape perturbation, particularly that induced by
cytoskeleton-disrupting drugs, alters (in certain circumstances) the activity
of specific signaling intermediates
(Schmid-Alliana et al., 1998;
Ren et al., 1999
;
Yujiri et al., 1999
;
Subbaramaiah et al., 2000
). A
pharmacological approach was selected to probe the involved pathways since
changes in cell structure may disrupt signaling `scaffolds' or interfere with
intracellular proteins, making data obtained with dominant-negative constructs
difficult to interpret. Several non-receptor tyrosine kinases, moreover, have
been implicated as upstream elements in signaling events that involve
cytoskeletal reorganization (Abram and
Courtneidge, 2000
; Schmitz et
al., 2000
). Consistent with these findings, the broad-spectrum
tyrosine kinase inhibitor genistein effectively blocked CD-induced PAI-1 mRNA
expression (>50% and 100% inhibition at 25 and 50 µM, respectively)
(Fig. 6).
pp60c-src, in particular, can be specifically activated by
actin network-modulating compounds (i.e. CD) in concentrations identical to
those required to initiate PAI-1 transcription
(Higgins et al., 1992
;
Lock et al., 1998
), suggesting
that src-family kinases may function in shape-dependent PAI-1 gene
regulation. Indeed, CD stimulated pp60c-src kinase
activity at least four-fold within 15 minutes of addition to quiescent R22
cultures (Fig. 7).
Preincubation of R22 cells with the more restrictive (src-family)
tyrosine kinase inhibitor PP1 markedly decreased CD-induced PAI-1 mRNA levels
(Fig. 6) and
pp60c-src activation
(Fig. 7). Herbimycin A produced
similar results in R22 (data not shown) as well as in NRK cells
(Hawks and Higgins, 1998
). PP1
was particularly effective (i.e. 50% inhibition of PAI-1 expression at 100 nM
and complete inhibition at 500 nM), strongly suggesting the participation of
src kinases in the inductive process
(Hanke et al., 1996
).
|
|
Involvement of ERK1/2 in CD-mediated PAI-1 gene
expression
Elements of the ras, raf, MAPK cascade associate with a
microfilament-linked signaling `particle'
(Carothers-Carraway et al.,
1999; Li et al.,
1999
), suggesting a cell structural basis for MAPK activation
similar to the regulation of Rho GTPase by the cytoskeleton
(Ren et al., 1999
). The
potential involvement of specific MAPKs as downstream mediators of
CD-initiated signaling was initially evaluated using PD98059 to inhibit
MEK-dependent signaling. Pretreatment of R22 cells with PD98059 significantly
reduced (at 10 µM) and virtually eliminated (at 25 µM) CD-stimulated
PAI-1 expression (Fig. 8).
CD-dependent ERK1/2 activation was confirmed using a coupled
immunoprecipitationin-vitro-kinase-assay in the presence of the
exogenous substrate, MBP. While there was some variation in CD-stimulated ERK
activity at early time points (i.e. 30 minutes) in replicate experiments,
total ERK-targeted MBP phosphorylation consistently increased four-fold by 1
hour and remained elevated for at least 2 hours
(Fig. 8). CD-responsive ERK
activation was approximately 50% that of serum-stimulated cells (standardized
to 10 minutes after FBS addition) and could be effectively suppressed (to
levels even below that of quiescent control cells) by PD98059
(Fig. 8). CD treatment
activated both ERK1 and ERK2 with only slight differences in kinetics
(Fig. 9). The level of
ERK1-directed MBP phosphorylation (at 2 hours) in response to CD approached
that of serum-stimulation; ERK2, by contrast, was more responsive to serum
than ERK1 in R22 cells (by at least two-fold) although the relative ability of
ERK1 and ERK2 to phosphorylate MBP in response to CD was similar
(Fig. 9). Total ERK1/2 protein
levels remained unchanged regardless of treatment conditions or relative
ERK1/2 activity. CD-mediated ERK1/2 activation coincided with the initial
increase in PAI-1 transcripts; both events, moreover, occurred in a delayed
and sustained fashion compared with the rapid and transient ERK1/2
phosphorylation response to serum.
|
|
To assess the relationships among signaling elements, pharmacological inhibitors that impacted on CD-mediated PAI-1 gene expression were evaluated for their ability to modulate ERK activity. Consistent with the requirements for PAI-1 induction (Figs 6 and 8), genistein and PP1 (and PD98059) completely inhibited CD-stimulated as well as basal ERK activity in the immunoprecipitation-in-vitro-kinase MBP assay, placing the PP1-sensitive kinases upstream of ERK (Fig. 10). The effect of these agents on PAI-1 expression/ERK activation profiles, moreover, was not simply caused by generalized toxicity or inhibition of CD-mediated microfilament disruption/cell-shape change. Neither CD or colchicine nor any of the pharmacological inhibitors used adversely affected cell viability over the time course used; PD98059 and PP1 also did not interfere with CD-initiated cytoskeletal rearrangements in R22 cells (data not shown). These data clearly position the PP1-sensitive kinase upstream of MEK and, more importantly, illustrate that cell-shape-dependent PAI-1 transcription is not merely a consequence of MEK-ERK autoactivation. Similar to the requirements for CD-dependent PAI-1 expression, colchicine-induced PAI-1 synthesis was significantly attenuated by pretreatment with PD98059, PP1 and puromycin. Inhibitor levels that ablated CD-mediated PAI-1 transcription (i.e., 25 µM PD98059, 170 nM PP1) were also sufficient to block both the colchicine-initiated PAI-1 response and ERK1/2 activation (Fig. 11).
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Discussion |
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src-family/MAP kinase signaling to PAI-1 transcription is not
unique to structural disruption in a specific cytoskeletal compartment, as CD
and colchicine were each efficient activators. It is quite possible that
altered dynamics in both the actin- and tubulin-based systems have common
downstream effectors. The similarity in signaling requirements between the two
stimuli (CD versus colchicine) used in the present paper to effect PAI-1
transcription, however, suggests that the associated changes in cell shape,
while the result of cytoskeletal network perturbation, may be a more likely
causative element in PAI-1 induction. CD- and colchicine-dependent PAI-1
expression, moreover, utilizes a tyrosine kinase/MEK-activated secondary
response pathway that is distinct from the primary mode of
growth-factor-induced PAI-1 transcription
(Ryan et al., 1996;
Kutz at al., 2001
).
TGF-ß1 transcription is similarly stimulated by CD
(Varedi et al., 1997
) and
TGF-ß1-induced PAI-1 expression is, at least partly, MEK dependent
(Kutz et al., 2001
). Although
this growth factor represents a likely secondary response intermediary in the
shape-initiated pathway of PAI-1 gene control
(Boehm et al., 1999
), the
present findings, clearly rule out an autocrine TGF-ß loop as a PAI-1
inductive mechanism.
Induced src-like kinase and MAPK activity as well as crosstalk
among the ras, rho, rac, cdc42 and related GTPase cascades may each
relate to cytoskeletal/cell-shape controls on PAI-1 gene regulation (this
paper) (Mucsi et al., 1996;
Afti et al., 1997; Schmid-Allina et al.,
1998
; Yujiri et al.,
1999
; Abram and Courtneidge,
2000
). CD stimulates rho GTPase function
(Ren et al., 1999
) which, in
turn, increases serum response factor (SRF) activity
(Hill et al., 1995
;
Alberts et al., 1998
) and
(potentially) rho-mediated PAI-1 transcription (Afti et al., 1997;
Park and Galper, 1999
;
Takeda et al., 2001
). Indeed,
the rho-GTPase-binding proteins mDia 1 and mDia 2 couple rho
GTPase and pp60c-src, with subsequent effects on cell
signaling, SRF activation and actin organization
(Tominaga et al., 2000
).
Microfilament disruption in response to CD, moreover, stimulates
pp60c-src kinase activity (this paper)
(Lock et al., 1998
), amplifies
tyrosine phosphorylation of Shc and promotes association of Shc with Grb2 as
well as adhesion-dependent ERK activation
(Barberis et al., 2000
). These
pathways may be relevant not only to shape-dependent regulation of the PAI-1
gene but also to its protease target uPA since a dominant-negative
src expression construct effectively attenuated luciferase reporter
expression driven by a uPA promoter sequences
(Irigoyen et al., 1997
;
Irigoyen and Nagamine, 1999
).
Previous findings suggest, moreover, that cytoskeletal controls on ERK
activity may proceed through two parallel but distinct (FAK- and
Shc-dependent) pathways, with src-family intermediates common to both
(Irigoyen and Nagamine, 1999
;
Barberis et al., 2000
).
Cytoskeletal disruption induces Shc phosphorylation and the formation of
Shc/Grb2 and Shc/FAK complexes; both FAK and pp60c-src are
required for optimal CD-initiated ras/ERK signaling. PAI-1 gene
induction is, indeed, sensitive to the tyrosine kinase inhibitor PP1, implying
that src kinases serve as upstream regulators of this pathway.
CD-stimulated matrix metalloproteinase expression in human dermal fibroblasts
is similarly a src-family kinase-dependent secondary-type response
(Lambert et al., 2001
). Recent
data, furthermore, indicate that p130cas enhances epidermal
growth-factor-dependent signaling events by acting as a substrate for
pp60c-src. pp60c-src phosphorylates
p130cas, promoting interactions with Grb2 or Shc/Shp2, activating
the ras/MEK/ERK cascade and inducing SRE-dependent gene transcription
(Hakak and Martin, 1999
). CD
does, in fact, stimulate a genistein-sensitive phosphorylation of
p130cas as well as formation of p130cas-FAK complexes
(R.S. and P.J.H., unpublished). These and previous findings
(Lock et al., 1998
; Hakek and
Martin, 1999; Barberis et al.,
2000
) suggest a model whereby cell shape changes initiate a
pp60src/p130cas-dependent signaling cascade
that results in MEK-ERK activation and PAI-1 expression.
The potential importance of morphology-linked controls on PAI-1
transcription is underscored by the marked increase in PAI-1 synthesis in
transformed, migrating, mechanical- and hypoxia-stressed as well as
growth-factor-stimulated cells (e.g.
Seebacher et al., 1992;
Eckstein and Bade, 1996
;
Ryan et al., 1996
;
Feng et al., 1999
;
Coats et al., 2000
;
Providence et al., 2000
;
Kutz et al., 2001
) and the
obvious changes in cytoskeletal dynamics associated with these processes.
Recent data suggest that sudden loss of cell-cell contact in cultured
epithelial cells by denudation injury stimulates PAI-1 mRNA/protein synthesis
specifically in the wound edge cohort but not in the distal, still
contact-inhibited, monolayer regions
(Providence et al., 2000
).
Since cadherin-dependent cell-cell contacts are sites of src kinase
localization (Calautti et al.,
1998
; Owens et al.,
2000
), one intriguing possibility is that loss of cell-to-cell
junction integrity, a common aspect of CD- and colchicine-induced cell body
retraction, may activate src kinases and downstream (MAPK) effectors
resulting in PAI-1 transcription.
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Acknowledgments |
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References |
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