|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 6 (September 15), 1999:
pp. 2029-2038
Fibrin Fragment Induction of Plasminogen Activator Inhibitor
Transcription Is Mediated by Activator Protein-1 Through a Highly
Conserved Element
By
Mitchell A. Olman,
James S. Hagood,
Warren L. Simmons,
Gerald M. Fuller,
Charles Vinson, and
Kimberly E. White
From the Department of Medicine, Division of Pulmonary and Critical
Care Medicine, and the Departments of Pathology, Pediatrics, and Cell
Biology, University of Alabama, Birmingham, AL; and the Laboratory of
Biochemistry, National Cancer Institute, Bethesda, MD.
 |
ABSTRACT |
Plasminogen activator inhibitor type-1 (PAI-1), a serine protease
inhibitor, affects the processes of fibrinolysis, wound healing, and
vascular remodeling. We have demonstrated that PAI-1 transcription is
induced by D dimer, a plasmin proteolytic fragment of fibrin,
supporting its role in negative feedback on peri-cellular proteolysis.
The focus of this study was to define the mechanism of D dimer's
effects on PAI-1 transcription. D dimer increased the binding activity
of the transcription factor activator protein-1 components c-fos/junD
and c-fos mRNA levels in a time- and concentration-dependent manner to
a greater extent than fibrinogen. Both basal and D dimer-induced PAI-1
transcriptional activity were entirely dependent on elements within the
 161 to 48 bp region of the PAI-1 gene in fibroblasts. Mutations within the AP-1-like element (59 to 52 bp) in the PAI-1 gene affected D dimer-induced transcriptional activity, c-fos/junD DNA binding, and basal and c-fos inducible PAI-1
transcriptional activity. Furthermore, expression of either wild-type
or mutant c-fos proteins augmented or diminished the response of the
PAI-1 promoter (161 to +26 bp) to D dimer, respectively. D
dimer-induced binding of c-fos/junD to the highly conserved and unique
AP-1 like element in the PAI-1 gene provides a mechanism whereby
specific fibrin fragments control fibrin persistence at sites of
inflammation, fibrosis, and neoplasia.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
PERSISTENCE OF CROSS-LINKED fibrin is a
frequent feature of the development of atherosclerosis, fibrosis, and
neoplasia. However, both the mechanism of its persistence and the
nature of its effect on specific cellular events remain largely
undefined.1-6 Type I plasminogen activator inhibitor
(PAI-1) is a 50,000-kD glycoprotein from the serpin superfamily of
genes.7,8 Like other serpins, PAI-1 forms protease
inhibitor complexes by suicide inactivation, with a second order rate
constant for inhibition of tissue type-plasminogen activator (t-PA) and
urokinase (u-PA) of approximately 107
mol/L 1s1.9,10 Its
physiologic roles include inhibition of fibrinolytic activity in
thrombi and extracellular matrix, as well as regulation of other
plasmin-dependent processes, including proteolytic and nonproteolytic
cell migration through an extracellular matrix and modulation of
plasmin-dependent transforming growth factor- (TGF-)
activation.11-13 PAI-1 participates in the biology of wound healing, lung injury/fibrosis, cancer metastasis, and atherosclerosis through modulation of fibrinolysis or cell adhesion.14-17
However, these complex in vivo processes share the common features of
PAI-1-expressing mesenchymal cells that adhere to, migrate through,
proliferate in, and/or actively remodel a fibrin-rich extracellular
matrix.4,18,19
Cellular expression of PAI-1 in fibroblasts can be modulated by
numerous inflammatory cytokines, including TGF, tumor necrosis factor- (TNF-), interleukin-1 (IL-1), and IL-6, as
well as by glucocorticoids.20-25 We have recently shown
that PAI-1 gene expression is upregulated in fibroblast-like cells in
vivo in a rodent model of lung fibrosis, in association with fibrinous
matrix deposition.4,26 We reasoned that the fibrin
deposition may not only reflect the consequences of PAI-1 inhibition of
PA-dependent fibrinolytic activity, but may directly participate in the
regulation of PAI-1 expression through a feedback loop mechanism. To
examine this possibility, terminal plasmin proteolytic fragments of
fibrin were purified and tested for their ability to induce PAI-1
expression in rat lung fibroblasts.27 The carboxyterminal
fragment, D dimer, induced PAI-1 transcription and expression to a
greater degree than the parent molecule, fibrinogen.27 The
focus of this study was to characterize the fibrin fragment-induced
trans-acting factors and their corresponding cis
elements within the regulatory region of the PAI-1 gene. Heterodimers
of c-fos/JunD heterodimers were increased in response to D dimer
stimulation and bound to the AP-1 like element (59 to 52
bp) of the PAI-1 promoter in a time-and concentration-dependent manner.
Moreover, this D dimer-induced c-fos expression was found to increase
PAI-1 transcriptional activation through this same element. The
promoter region encompassing this functional element is not present in
other known promoters, yet it exhibits strict evolutionary conservation
at the nucleotide level among mammalian species (97%
identity).28 These findings suggest a key role for this
regulatory site in the molecular pathways underlying fibrin dissolution
and cell adhesion during the processes of angiogenesis,
atherosclerosis, and fibrosis.
| |
MATERIALS AND METHODS |
Materials.
The 2,400-bp 5' flanking region of the rat PAI-1 gene was kindly
provided by Dr T. Gelehrter (University of Michigan, Ann Arbor,
MI).28,29 Promoterless firefly and Renilla
luciferase expression plasmids (pGL2 Basic, pRL-TK) and all reagents
for cell lysate luciferase assay were obtained from Promega Corp
(Madison, WI). The cytomegalovirus (CMV) expression vector pcDNA3 was
obtained from Invitrogen (San Diego, CA). All antibodies to fos/jun
family members were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The rat lung fibroblast cell line (RLF-6) was obtained from ATCC
(Bethesda, MD). Cationic lipid transfection reagent (lipofectamine; 3:1
DOSMA:DOPE), serum-free transfection media (Optimem), and HEPES
(N-2-hydroxyethylpiperazine-N'-2-ethane sulfonic acid) were obtained from Life Technologies (Gaithersburg, MD). Human fibrinogen and human factor XIII were obtained from Calbiochem (San Diego, CA).
Plasmin was obtained from American Diagnostica (Greenwich, CT). Lysine
and gelatin sepharose were obtained from Pharmacia Biotech Inc
(Piscataway, NJ). The cell propagation media (Ham's F-12) were
obtained from Mediatech (Herndon, VA). Chemical reagents were obtained
from Sigma Chemical Co (St Louis, MO). Pathogen-free, male F344 rats
were obtained from Charles River Labs (Wilmington, MA). Purified c-fos
and JunD proteins and mammalian expression vectors encoding the acidic
fos mutant (A-fos) were provided by Dr C. Vinson (Laboratory of
Biochemistry, National Cancer Institute, Bethesda, MD).30
Cell isolation and propagation.
Animal protocols used for tissue harvesting were approved by the
Institutional Animal Care and Use Committee (#9502067) of the
University of Alabama at Birmingham (Birmingham, AL). Primary cultures
of rat lung fibroblasts (F344) were established from 6-week-old F344
rats by enzymatic digestion and mechanical disaggregation as previously
described.27 Fibroblasts were selected by differential adherence to tissue culture plastic and maintained at 37°C in humidified 5% CO2 in minimal essential media supplemented
with 10% fetal bovine serum (FBS), 20 mmol/L HEPES, and
penicillin/streptomycin/gentamycin. Cultures were determined to be free
of Mycoplasma contamination by polymerase chain reaction (PCR) using
Mycoplasma-specific probes as described (Stratagene, La Jolla, CA).
Cell identity of primary cultures was verified by the absence of tight
junctions, by production of collagen types I and III, and by typical
morphology on phase contrast and electron microscopy.27
Confluent primary cultures (less than passage number 5) were made
quiescent by placement in 0.4% serum for 48 hours before stimulation
with and without the indicated agonists in serum-free media. The rat
lung fibroblast nontransformed cell line (RLF-6) was grown to
confluence in Ham's F-12 containing 20% FBS as recommended by ATCC.
Preliminary experiments showed a PAI-1 induction in the RLF-6 cell line
similar to that reported by our laboratory for primary
cultures.27
Isolation and preparation of purified plasmin proteolytic fragments
of cross-linked fibrin.
Fibrin(ogen) fragments were prepared and purified as published by the
investigators as modified from previous methods.27,31-34 Separation and purification steps were validated by Coomassie staining
of electrophoresed pooled fractions on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and by Western blotting with thrombin, fibronectin, plasminogen, fibrin fragment E, and D domain specific antibodies, as
published.31
Preparation of PAI-1 gene-Luciferase expression constructs.
Segments of the rat PAI-1 gene 5' flanking region were cut
(5'-p928-Sma I, p280-Kpn I, p161-Eco72I,
3' +26-Xho I) and ligated to the Luciferase expression
plasmid, pGL2-Basic (Promega). The 5' deletional construct at
48 to +26 (p48) was generated by PCR using 5' primers
complementary to the PAI-1 gene sequence. Mutations at the putative
AP-1 site (p161wt; 59 to 52 site; TGAGTTCA) to the
nonbinding mutant site (p161mut; TGTGTTTG) or to the
consensus AP-1 binding sequence (p161con; TGAGTCA) were performed by
single-strand mutagenesis using commercially available reagents
(QuikChange Mutagenesis kit; Stratagene, San Diego, CA) according to
the manufacturer's instructions (Fig 1).
Briefly, mutated constructs were generated using the wild-type
double-stranded p161-Luciferase as a template. The mutated
single-stranded primers were annealed to the template, followed by
first-strand synthesis using thermostable Pfu I DNA polymerase
(16 cycles of 95°C, 55°C, and 68°C), digestion of the
wild-type template with Dpn I, and transformation of the
resultant DNA into competent cells (Epicurean Coli-XL2). All constructs
were verified by automated DNA sequencing as performed by the
University of Alabama at Birmingham Core DNA Sequencing Facility.
View larger version (35K):
[in this window]
[in a new window]
| Fig 1.
Effect of 5' deletion of the PAI-1 gene on basal
and D dimer-inducible PAI-1 transcriptional activity. Progressive
deletions of the 5' flanking region of the PAI-1 gene were
generated by restriction digestion or PCR and ligated to a
luciferase-encoding reporter plasmid. The plasmids were transiently
transfected into rat lung fibroblasts along with the thymidine kinase
promoter-Renilla luciferase encoding plasmid. For determination of
promoter activity, cells were incubated for 48 hours posttransfection
in 0.4% serum containing media followed by either serum-free media
([A], right, basal activity) or SFM plus D dimer (1.1 µmol/L;
[B], inducible activity) for 6 hours. Raw data are tabulated as the
ratio of firefly luciferase activity to that of its Renilla
luciferase activity (mean ± SD) and reported relative to the value
for the p928 plasmid (assigned value of 100). *P < .05 relative to the p928 values. +P < .05 relative to the
p161wt values by ANOVA/Newman-Keuls test. (A) Relative basal activity.
Constructs are as indicated at left. Mutations were performed at the
AP-1-like site (59 to 52) of the PAI-1 gene with otherwise
intact 161 to +26. PAIcon marks the TGAGTCA element, PAIwt marks
the TGAGTTCA element, and PAImut marks the
TGTGTTTG element at the 59 to 52
positions of p161. Underscored letters denote deviations
from the consensus AP-1 element. (B) D dimer-inducible transcriptional
activity. Transfected plasmids are as indicated.
|
|
Transient transfection of rat lung fibroblasts.
Transient transfection of a nontransformed cell line of rat lung
fibroblasts (RLF-6; 3.5 × 105 cells in
35-cm2 wells) was performed with cationic liposomes (6 µg/1.5 µg DNA; Lipofectamine). Cells were plated at the above-noted
density in Ham's F-12/20% FBS, washed twice with Optimem, and
incubated with the lipid/DNA complexes for 6 hours in Optimem. At this
point, the cells were incubated with Ham's F12 with 0.4% FBS or
Ham's F12 containing D dimer (1 µmol/L) for 48 hours. Alternatively, the cells were transfected incubated in Ham's F12 containing 0.4% serum for 48 hours followed by either serum-free media or D dimer (1 µmol/L) in serum-free media for 6 hours. The full coding regions of
wild-type rat c-fos (c-fos) and the c-fos mutant (fosBR; basic DNA
binding region deletion; kindly provided by Dr T. Townes, University of
Alabama at Birmingham) were ligated into the mammalian cell expression
plasmid, pcDNA3, downstream of the CMV early enhancer-promoter region
and a 3' bovine growth hormone polyadenylation signal. The
indicated PAI-1 promoter-luciferase plasmids (1 µg/well) were transfected along with either c-fos, fosBR, or acidic fos (Afos; acidic
amino acid substituted DNA binding region) expression plasmids (0.15 µg/well) and a Renilla luciferase encoding plasmid (pTK-RL; 0.15 µg/well) as a standard for transfection efficiency in Lipofectamine (5 µg/well). Firefly luciferase activity was assayed in cell lysates (25 mmol/L Tris-phosphate, pH 7.8, 1% TX-100, 2 mmol/L dithiothreitol [DTT], 2 mmol/L
1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid, 10%
glycerol) in the presence of coenzyme A, ATP, and the substrate luciferin. Renilla luciferase activity was assayed sequentially in the
same sample by quenching the firefly luciferase activity and adding the
Renilla luciferase specific substrate, coelenterazine, according to the
manufacturer's instructions (Promega). Light units were measured on a
Luminometer (Turner Model 20e; Promega), and data are expressed as the
ratio of firefly luciferase activity to that of Renilla luciferase to
account for variability in transfection efficiency among wells and
conditions. Preliminary experiments using cell lysates demonstrate
linearity (r2 = .998) and reproducibiity (CV = 3%)
of the luciferase assays over 6 logs in the range of our measurements.
Luciferase activity values of greater than 3 standard deviations above
background were accepted.
Electrophoretic mobility shift assay (EMSA).
Primary rat lung fibroblasts were exposed to D dimer (at the indicated
concentrations in Dulbecco's modified Eagle's medium [DMEM]) or serum-free DMEM for 15 to 60 minutes, and
nuclear extracts were prepared, as described.35,36 Washed
cells (106 to 107) were pelleted in
phosphate-buffered saline (PBS; 4°C) and lysed by repeated
pipetting at 4°C (in 10 mmol/L HEPES, pH 7.9, 1.5 mmol/L
MgCl2, 10 mmol/L KCL, 0.5 mmol/L DTT, and 0.2 mmol/L
phenylmethyl sulfonyl fluoride [PMSF]). Nuclear proteins were
extracted (420 mmol/L NaCl, 20 mmol/L HEPES, pH 7.9, 1.5 mmol/L
MgCl2, 0.5 mmol/L DTT, and 0.2 mmol/L PMSF) and total
protein was measured by commercially available methods (BCA method;
Pierce, Rockford, IL). Oligonucleotides used in EMSA assays were (1)
PAIcon, a 21 mer consisting of the AP-1 consensus region (TGAGTCA)
flanked by 14 bp from the PAI-1 gene (66 to 60 and
51 to 45); (2) PAIwt, a 21 mer composed of the wild-type
PAI-1 sequence (TGAGTTCA; 60 to 52) plus flanking
sequence (66 to 45); or (3) PAImut, a 21 mer composed of
a mutated AP-1 site (TGTGTTTG) flanked by the
PAI-1 sequence (66 to 60 and 51 to 45; bold
denotes variation from the consensus AP-1). Oligonucleotides were
labeled with [-32P]-nucleotide using the Klenow
filling reaction, resulting in a specific activity of at least
106 cpm/µg DNA. The labeled oligonucleotide (20,000 cpm)
was allowed to combine with 1 µg of nuclear protein for 20 minutes at
25°C (10 mmol/L HEPES, 0.1 mmol/L EDTA, 10% glycerol, 100 mmol/L
KCl, 200 µg/mL bovine serum albumin [BSA], 1 mmol/L DTT, and 0.2 mmol/L PMSF) in the presence of the nonspecific competitor poly dI-dC (100 µg/mL). For specific competition experiments, a varying molar excess (up to 100-fold) of cold unlabeled oligonucleotide was added to
the labeled probe before binding reaction. For immunological identification of the transcription factor(s), factor-specific antibodies (1 to 1,000 ng/reaction; Santa Cruz
Biotechnologies, Santa Cruz, CA) were added to the binding reaction (30 minutes at 4°C) before the addition of the oligonucleotide. In all
cases, the entire reactions (20 µL) were electrophoresed under
nondenaturing conditions (4.8% to 6% acrylamide/0.5× TBE at 500 V for 1 hour at 4°C), followed by autoradiography. Band
radioactivity was quantified using a phosphorimager (Molecular
Dynamics, Sunnyvale, CA), and autoradiograms were made for presentation
in light tight cassettes with intensifier screens at 70°C
(Kodak XAR film; Eastman Kodak, Rochester, NY).
Northern blot analysis.
Total RNA was isolated from rat lung fibroblasts under the indicated
conditions and analyzed by Northern blotting as previously published by
our laboratory.4,27 Band density was quantified using a
phosphorimager, and data are presented as the ratio of the c-fos mRNA
specific band to that of the 18S rRNA band to normalize for loading and
transfer. Autoradiograms were made for presentation in light tight
cassettes with intensifier screens at 70°C (Kodak XAR film).
Statistical analysis.
Interval data were compared by means of an ANOVA.37 Where
differences were detected, a Dunnett's test or Newman-Keuls multiple comparisons procedures were used to test for differences in inducible firefly/Renilla luciferase activities between specific promoter constructs or in c-fos mRNA induction or AP-1 binding activity under
specific conditions.37 Results are expressed as the mean ± standard deviation unless otherwise indicated. Significance was
accepted at the P < .05 level for all analyses.37
| |
RESULTS |
Determination of the critical DNA regions for basal and D
dimer-inducible PAI-1 transcriptional activity.
To determine the basal and D dimer-responsive elements from the
5' flanking region of the PAI-1 gene, we performed transient transfections with several PAI-1 promoter-luciferase reporter constructs in rat lung fibroblasts. Upon progressive 5' deletion of the PAI-1 promoter, the basal activity first increased slightly (p928 v p280) and then decreased 75-fold (p928 v p48;
Fig 1A). Furthermore, upon mutation at the AP-1-like element at
59 to 52 bp (p161wt to p161mut), the basal PAI-1 promoter
activity was reduced to approximately one third of its wild-type
activity, whereas replacement of the same element with that of the
consensus AP-1 sequence (p161con) increased the PAI-1 promoter activity by 50% greater than that of the wild-type (Fig 1A).
D dimer directly increased transcription of PAI-1 mRNA, as measured by
nuclear run-off assay, and similarly enhanced PAI-1 promoter-driven
luciferase activity 4.5- ± 1-fold (using p928) in rat lung
fibroblasts. Relative to the largest promoter fragment tested, D
dimer's effect was preserved in the wild-type 161 to 48
region (p161) but was reduced by two thirds upon mutation of the
AP-1-like element from the same region (59 to 52 bp; p161mut) and lost upon deletion of this region (p48; Fig 1B). In
support of the importance of the AP-1-like element, D dimer's effect
was slightly enhanced by the presence of the consensus AP-1-containing
element (in p161con; Fig 1B). We also noted a blunting of D dimer's
effect using the p280 construct (280 to +26 bp), but its
restoration upon further 5' deletion (to p161wt; Fig 1B). In
summary, these data indicate that the precise sequence at the
AP-1-like element at 59 to 52 regulates both basal and D
dimer-inducible PAI-1 promoter activity.
Time and concentration dependence of D dimer-induced DNA-protein
binding.
Having demonstrated the AP-1-like element at 59 to 52 of
the PAI-1 gene was functionally relevant, we sought to characterize the
time and dose response of nuclear protein binding to D dimer initially
by an oligonucleotide containing a known AP-1 consensus element
(PAIcon). Rat lung fibroblasts were incubated with highly purified
fibrin D dimer, and nuclear extracts were assayed by EMSA. Band density
was quantified by densitometry. D dimer exposure resulted in a rapid,
transient, 5-fold increase in binding activity to PAIcon
oligonucleotide (PAIcon; 66 to 46; with consensus AP-1
site at 59 to 53; TGAGTCA) that was concentration
dependent (n = 5/condition; P < .01). This activity peaked at
45 minutes after exposure to 1 µmol/L D dimer and was noted with as
little as as little as 50 nmol/L D dimer, with the curve plateau at 1 µmol/L D dimer after 45 minutes. Although this activity was similarly demonstrable with an oligonucleotide consisting of the wild-type PAI-1
gene sequence (PAIwt; wt 66 to 45; 59 to 52
is TGAGTTCA), it was sequence specific. The DNA-protein complex
B was effectively competed by 100-fold excess of unlabeled
oligonucleotide, either PAIcon or PAIwt, but not a mutated PAI gene
oligonucleotide PAImut (PAImut; wt 66 to 45; 59 to
52 is TGTGTTTG; see
Fig 2B), and the DNA-protein complex formed
with the PAIcon or PAIwt oligonucleotides had the same migration
pattern, whereas there were no retarded bands with the free probe
control (Fig 2B). Because the DNA element within the PAI-1 gene
(59 to 52) varies from the AP-1 consensus by a 1-bp
insert (consensus, TGAGTCA; PAI-1 gene, TGAGTTCA), we compared
the in vitro affinity of the induced nuclear proteins towards the
consensus AP-1 sequence, the AP-1 like sequence in the wild-type PAI-1
gene, and a triple AP-1 site mutant (TGTGTTTG; PAImut)
by competition EMSA. Quantitative cross-competition analysis for
binding of the D dimer-induced nuclear proteins to
32P-labeled PAIcon showed a 50-fold difference in apparent
affinity to the wild-type sequence relative to that of the consensus
sequence (Fig 2A [ and  ] and B). In contrast, the triply
mutated oligonucleotide (PAImut) did not effectively compete for
binding (Fig 2A [ ] and B).
View larger version (28K):
[in this window]
[in a new window]
| Fig 2.
Relative affinity of D dimer-induced proteins for the
PAI-1 AP-1-like sequence and the AP-1 consensus sequence. Nuclear
proteins from D dimer-exposed fibroblasts (1 µmol/L for 45 minutes)
were analyzed by EMSA with a 32P-PAIcon oligonucleotide
(66 to 45, del-55; TGAGTCA). The indicated molar excesses of
unlabeled PAIcon, PAIwt (TGAGTTCA; PAIwt) or PAImut
(TGTGTTTG) oligonucleotides were added to the
binding reactions, and the reactions were analyzed by EMSA. Band
densities of the retarded B complexes were quantified on a
phosphorimager. (A) Quantitative competition of protein binding to
32P-labeled PAIcon by unlabeled PAIcon, PAIwt, and PAImut.
Data are plotted as band radioactivity (mean + SE) relative to that
with no unlabeled PAIcon. (B) Representative EMSA autoradiogram. Lane
1, free labeled PAIcon probe; lanes 2 through 5, labeled PAIcon with
indicated molar excesses of unlabeled PAIcon added to the protein
binding reaction; lanes 6 through 9, labeled PAIcon with indicated
molar excesses of unlabeled PAIwt; and lanes 10 and 11, labeled PAIcon
with indicated molar excess of unlabeled PAImut.
|
|
Identification of the components of the D dimer-inducible DNA-protein
complex.
To identify the transcription factor(s) that was induced to bind to the
functional AP-1-like element (66 to 45) in the PAI-1 gene upon D dimer exposure, EMSAs were performed on the same nuclear extracts in the presence of factor-specific antibodies. Direct competition and/or supershifting of complex B was noted with antibodies to both fos and jun family proteins, but not those raised against the
irrelevant antibody CD-11b (32P-PAIcon oligo;
Fig 3A). This pattern of
competition/supershifting strongly suggests that members of the AP-1
family are induced to bind by D dimer. A more detailed analysis using
antibodies specific to individual fos/jun proteins indicates that c-fos
and junD are major components of D dimer-induced fos/jun proteins in
complex B, whereas fra-2 and junB appear to play a minor role (32P-PAIcon; Fig 3B). Identical fos/jun family members bind
to the PAI-1 wild-type oligonucleotide (32P-PAIwt oligo;
Fig 3C) as the consensus. Taken together, these observations indicate
that D dimer fragment will rapidly induce the DNA binding activity of
c-fos/junD heterodimers in rat lung fibroblasts.
View larger version (45K):
[in this window]
[in a new window]
| Fig 3.
Immunologic identification of D dimer-induced DNA binding
proteins. Subconfluent rat lung fibroblasts were incubated in 0.4%
serum for 48 hours followed by exposure to D dimer (1 µmol/L for 45 minutes) or serum-free media controls (SFM). Nuclear proteins were
extracted and analyzed by EMSA with 32P-PAIcon (A and B;
PAIcon) or 32P-PAIwt (C) as indicated below the
autoradiograms. The effect of antibodies that are broadly
cross-reactive to fos/jun proteins and to specific fos/jun family
members on the protein-DNA binding was assessed through an examination
of the competition or supershifting of complex B in the EMSA
autoradiogram. (A) Increasing titer of fos (lanes 3 through 6) and jun
(lanes 7 through 10) family antibodies from 1 to 2,000 ng/binding
reaction. Antibody to CD11b serves as an irrelevant control antibody.
(B) Left autoradiogram, indicated antibodies to specific fos family
members, with negative control (CREB-2 Ab). Right autoradiogram,
indicated antibodies to specific fos family members, with negative
control (ATF-2 Ab). (C) Indicated antibodies to specific fos and jun
family members, with 32P-labeled PAIwt.
|
|
The AP-1 dependence of PAI-1 promoter activity is sequence specific
and a function of c-fos/JunD availability.
To determine the functional significance of the AP-1 increases noted in
response to D dimer, wild-type and altered PAI-1 promoter fragments
were tested for their c-fos dependence in cotransfection assays.
Expression of wild-type c-fos augmented both basal (SFM or 0.4% serum)
and D dimer-inducible PAI-1 promoter activity in a sequence-specific
manner. Upon triple mutation (p161mut) or deletion (p48) of the
AP-1-like element, there was a 3- and 100-fold loss of basal
transcriptional activity, respectively, as well as a loss of c-fos
inducibility (Fig 4A). A
comparison of the c-fos inducibility seen with consensus AP-1
sequence-containing promoter (p161con) with that of the wild-type
(p161wt) showed an increased dependence of the consensus-containing
promoter (p161con) to c-fos by 2-fold (Fig 4A).
View larger version (45K):
[in this window]
[in a new window]
| Fig 4.
Effect of AP-1 protein overexpression on basal
and D dimer-inducible PAI-1 transcriptional activating activity.
Subconfluent rat lung fibroblasts were cotransfected with plasmids
expressing either wild-type c-fos (c-fos) or JunD (JunD), a
nonfunctional mutant c-fos (DNA-binding basic region deleted; fos BR),
or a peptide containing an acidic amino acid-substituted DNA binding
region (A-fos) along with the indicated PAI-1 promoter-luciferase
reporter constructs and the transfection control Renilla
luciferase reporter plasmid. p161con, p161wt, p161mut, and p48
PAI-1 promoter-luciferase constructs are as defined in Fig 1. Results
are plotted as the ratio of firefly/Renilla luciferase
activities (mean + SE) relative to the activity ratio seen with the
empty vector. (A) Sequence specificity of c-fos induction. ( ) Empty
vector; ( ) fos BR vector; () c-fos vector. *P < .05 relative to the mutant fos values. (B) Effect of dominant negative
c-fos (A-fos) on D dimer inducibility of p161wt. Fibroblasts were
transfected as described above and exposed to 0.4% serum-containing
media for 48 hours followed by either D dimer (1 µmol/L) or
serum-free media for 6 hours. () Empty vector; () A-fos vector;
() JunD or JunD/c-fos vectors. *P < .05 relative to the
empty vector. +P < .05 relative to the c-fos vector by
ANOVA/Newman-Keuls test.
|
|
To define the significance of c-fos and JunD to D dimer's effect on
PAI-1 promoter activity, fibroblasts were cotransfected with the p161
reporter construct and either a wild-type c-fos expressing vector, an
empty vector, or an acidic mutant fos expressing vector and incubated
with D dimer. Preliminary experiments documented the capacity of a
2-fold molar excess of the acidic mutant fos protein to block 99% of
c-fos/JunD binding to the wild-type PAI-1 oligonucleotide (PAIwt;
66 to 45 bp) by EMSA (data not shown). Expression of
wild-type c-fos (c-fos) or JunD (junD) enhanced the capacity of the
cell to respond to D dimer with an increase in PAI-1-promoter
activity, whereas transfection of the mutant fos (A-fos) diminished the
capacity of the cell to respond to D dimer (Fig 4B). These data
indicate that manipulation of the availability of c-fos and junD to
bind to DNA will alter the basal and D dimer responsiveness of the
PAI-1 promoter in a sequence-specific manner.
Relative induction of c-fos by D dimer and its parent molecule
fibrinogen.
Because we have shown in prior work that PAI-1 expression was greater
in fibroblasts incubated with D dimer than with fibrinogen, the effects
of fibrinogen and D dimer on AP-1 element binding activity and mRNA
levels for c-fos and Jun D were similarly compared. Both proteins
induced DNA binding activity to both the wild-type oligonucleotide
(PAIwt) and the AP-1 consensus-bearing oligonucleotide (PAIcon) by EMSA relative to serum-free media after 45 minutes (n = 3/condition; P = .0002, ANOVA;
Fig 5, complex B: left, PAIcon; right,
PAIwt). However, the increase in DNA binding activity in cells exposed
to D dimer was 2.5-fold greater than that of the fibrinogen (P < .05) and equivalent to that of 10% serum-exposed cells (Fig 5).
Similarly, whereas both proteins increased c-fos mRNA steady-state
levels, the peak D dimer response was 4-fold greater in magnitude (n = 3/condition; P = .001, ANOVA) and tended to last longer
(P = .08) than that of fibrinogen
(Fig 6A and B). JunD mRNA, in contrast, was
not induced by either protein (data not shown). This quantitation was
performed by normalizing the c-fos or JunD mRNA band density of each
lane with that of the 18S RNA to account for any variability in loading
and transfer. In summary, the differences between D dimer and
fibrinogen-induced AP-1 responses in the fibroblasts were largely in
the magnitude of the response in c-fos mRNA.
View larger version (55K):
[in this window]
[in a new window]
| Fig 5.
Comparison of fibrinogen and D dimer induction of
AP-1-DNA binding activity by EMSA. Fibroblasts were incubated with
either fibrinogen or D dimer (1 µmol/L) for 45 minutes in serum-free
media, followed by harvesting of nuclear extracts, which were analyzed
by EMSA using a 32P-labeled PAIcon (A, left) or PAIwt (A,
right) oligonucleotides. Complex B band density was quantified on a
phosphorimager and plotted as mean +SE (n = 3/condition) in (B).
Serum-free media or serum (10%) -containing media-exposed cells served
as negative and positive controls, respectively. DDex, FGNex, 10%Sex,
and SFMex denote nuclear extracts from D dimer, fibrinogen, 10%
serum-containing media, and serum-free media-exposed cells,
respectively. 100X PAIcon denotes inclusion of a 100-fold molar excess
of unlabeled PAIcon oligonucleotides in the binding reaction. (A) Left,
EMSA using the 32P-labeled PAIcon oligonucleotide; right,
EMSA using the 32P-labeled PAIwt oligonucleotide. Complex B
is indicated. (B) Quantitative presentation of complex B band density
(AP-1). () Data from 32P-labeled PAIcon oligonucleotide;
() 32P-labeled PAIwt oligonucleotide.
|
|
View larger version (27K):
[in this window]
[in a new window]
| Fig 6.
Induction of c-fos mRNA by D dimer. Subconfluent rat lung
fibroblasts were incubated in 0.4% serum for 48 hours followed by
incubation with either fibrinogen or D dimer (1 µmol/L) in serum-free
media for 15 to 60 minutes as indicated. Northern blotting of the RNA
was performed as described in Materials and Methods. Autoradiogram
bands were analyzed by phosphorimagery. Data are plotted as the mean + SE of the band density relative to that of SFM alone (n = 3/time
point). (A) Representative autoradiogram. SFM, FBS, FGN, and D dimer
denote the mRNA from serum-free media, 10% fetal bovine serum,
fibrinogen, and D dimer-exposed cells, respectively. (B) Fold induction
of c-fos/18S RNA (loading and transfer control) band intensity values
relative to that of cells incubated in serum-free media. ()
Fibrinogen-exposed cells; () D dimer-exposed cells.
|
|
| |
DISCUSSION |
The major finding in this study is the identification of the AP-1-like
DNA element as an important transcriptional control element in the
PAI-1 gene. In support of our key observation, deletion or mutation of
this AP-1-like element significantly affects the level of basal
transcription and D dimer responsiveness of a PAI-1 promoter-luciferase
reporter construct. Furthermore, the basal transcription and D dimer
responsiveness of the PAI-1 promoter is altered in a sequence-specific
manner by manipulation of intracellular AP-1 levels in cotransfection
assays. Our analysis of AP-1 family members in D dimer exposed cells
indicates that D dimer enhances c-fos/junD DNA binding activity to the
AP-1-like element (59 to 52) in a dose- and
time-dependent manner. We have demonstrated the specificity of this
binding activity by competition EMSA by varying both the nucleotide
sequence and by using antibodies to specific fos/jun family members.
Furthermore, altering the levels of fos/jun resulted in detectable
changes in PAI-1 promoter activity, which was similarly nucleotide
sequence specific. Taken together, these data suggest that, in response
to D dimer stimulation, AP-1 activity increases to enhance PAI-1
transcription through its unique AP-1-like element at 59 to
52 in the PAI-1 promoter.
The PAI-1 gene sequence of the rat, mouse, and human species are
strikingly similar (97% homologous) in the region from 60 to
the TATA box (31), with identical AP-1-like element sequences, whereas the upstream region (928 to 60) is significantly
less homologous (70%).28 Furthermore, this proximal region
appears to be entirely unique among known promoters in the NCBI
database, and this same element has been implicated in the PAI-1
transcriptional response to TGF- and to phorbol myristate acetate
(PMA) in several cell types.38-40 This
suggests that the elements within this proximal region of the PAI-1
promoter participate in cell responses that are both unique and
critical to PAI-1 physiology. One possible function of this highly
conserved proximal region may be to coordinate the AP-1-dependent
expression of PAI-1 with the cell cycle.41-46 In this
context, PAI-1 protein may participate in cell cycle regulation by
modulating the integrity of cell-matrix contacts, either through its
matrix proteolytic inhibitory action and/or through blocking of cell
surface receptor-ligation sites on matrix
vitronectin.14,15,47
The AP-1 response to D dimer, as characterized, is both specific and
physiologically relevant. First, the D dimer fragments used were shown
to be free of other PAI-1 regulatory molecules, including plasmin,
thrombin, fibronectin, t-PA, and TGF-, by immunoblotting. Second,
the magnitude of the increase in AP-1 binding activity and c-fos mRNA
in response to D dimer was 3 times that of equimolar amounts of
fibrinogen. D dimer was also a more potent inducer (3-fold) of AP-1
binding activity than equimolar fibrinogen fragment E (1 µmol/L) or
0.4% serum (280 µg/mL protein; not shown), thereby ruling out a
nonspecific protein effect. Interestingly, the relative induction of
c-fos mRNA and AP-1-DNA binding activity in response to D dimer versus
fibrinogen corresponds to their relative potency of induction of PAI-1
protein and mRNA.27 This suggests that D dimer possesses
conformationally dependent AP-1 signaling epitopes that regulate PAI-1
expression. These epitopes may be exposed upon plasmin cleavage of
cross-linked fibrin in a manner similar to that seen with
thrombin-cleavage of fibrinogen. The thrombin-cleaved fibrinogen chain possesses heparin-binding domains important for its binding to
endothelial cells.48,49 Potential D dimer conformational
epitopes have yet to be characterized, despite that fact that it is the
major soluble fibrin-derived molecule in tissue during the
matrix-remodeling process seen after inflammatory injury or
thrombosis.26,50,51
Under the conditions of the competitive EMSA, it appears that the
affinity of the inducible AP-1 for the PAI-1 wild-type promoter AP-1-like element (TGAGTTCA) is lower than that for the consensus element (TGAGTCA). However, it is apparent that the weak interactions at the wild-type element can result in physiologically significant transcriptional responses. In support of this contention, the level of
c-fos-inducible PAI-1 promoter activity within the wild-type construct
(p161wt) or the consensus construct (p161con) differed by only 2-fold,
and the basal PAI-1 promoter activity was approximately equivalent
(Figs 1, 2, and 4), whereas the nonbinding mutant promoter activity was
one third less and was not inducible upon c-fos cotransfection. This
apparent difference in relative affinity and transcriptional activating
activity may reflect the importance of the state of activation of fos,
of interactive effects with coactivators, of DNA conformational
effects, or of redundancy among AP-1 family members.52-54
Several lines of evidence point towards c-fos/junD as the AP-1 member
of greatest significance. Namely, c-fos/junD are the most prominent
AP-1 family members present in D dimer exposed cells by EMSA, the
effect of cotransfection of c-fos is dependent on the sequence at the
AP-1-like element (59 to 52), and cotransfection of
A-fos, which will bind to Jun protein, thereby blocking Jun-related AP-1 transcriptional activity, partially abrogated the PAI-1 promoter response to D dimer. Additional indirect evidence is that the relative
potency of c-fos induction by D dimer and fibrinogen parallels that of
their PAI-1 inducing capacity.27 Because EMSA is a
semiquantitative technique, it remains possible that quantitatively minor, or as yet unknown, AP-1 family members participate in the transcriptional response.
The persistence of extracellular matrix fibrin in disease states,
including fibrosis, neoplasia, and atherosclerosis, demonstrates the
consequences of blocked fibrinolysis in these disorders, and recent
studies in mice that are deficient in plasminogen and/or fibrinogen
illustrate the basic biological significance of fibrin/fibrinolysis to
matrix remodeling.1,5,26,51,55 Our model suggests a mechanism whereby the fibroblast may control peri-cellular proteolysis of its matrix environment through AP-1-mediated PAI-1 induction (Fig 7). A PAI-1-inductive signal is initiated by
fibrin fragments that are the product of plasminogen
activator-dependent plasmin action and are mediated by AP-1-dependent
transcriptional activation of the PAI-1 gene. The PAI-1 produced will
block further fibrinolysis (D dimer formation) through PAI-1's
inhibitory action on plasminogen activation, thereby closing a negative
regulatory feedback loop. This feedback of proteolytically derived
matrix fragments on peri-cellular fibrinolytic activity may underlie
the cellular sensing and maintenance of extracelluar matrix integrity
in both normal and disease states.
View larger version (20K):
[in this window]
[in a new window]
| Fig 7.
Model of D dimer-initiated negative regulatory feedback
loop of peri-cellular fibrinolysis. As a consequence of plasmin
proteolysis of cross-linked fibrin, D dimer is generated and initiates
a signal that results in AP-1-dependent induction of PAI-1
transcription using the 59 to 52 element. New PAI-1 protein is
synthesized and, upon secretion, binds with high affinity to
plasminogen activators, thereby blocking further activation of
plasminogen to active plasmin. Upon loss of plasmin action,
fibrinolysis ceases, allowing fibrin persistence and halting further
generation of D dimer.
|
|
In summary, we have demonstrated the dependence of basal, fibrin D
dimer and fibrinogen-stimulated PAI-1 transcription on an AP-1-like
element unique to the PAI-1 gene. At least 2 proteins that comprise the
heterodimeric transcription factor, AP-1, after D dimer stimulation are
c-fos and junD. Although the affinity of the induced protein complex
for the AP-1-like element in the PAI-1 promoter varied considerably
from its affinity to a consensus AP-1 element, dependence of PAI-1
transcription of the wild-type sequence on c-fos expression was easily
demonstrated. The strict evolutionary conservation of this sequence
underscores the importance of its role in regulating the expression of
PAI-1, a molecule essential to the maintenance of fibrin and matrix
homeostasis in disease states.
| |
ACKNOWLEDGMENT |
The authors acknowledge Patrick Miller, Dr Wenqi Jiang, and Dr James J. Marsh for their technical assistance and provision of reagents.
| |
FOOTNOTES |
Submitted February 2, 1999; accepted May 20, 1999.
M.A.O. was a Parker B. Francis Research Foundation fellow during part
of this work. This work was supported by grants from the American Lung
Association, the American Federation of Clinical Research, the Veterans
Administration MERIT Review, the National Institutes of Health to
M.A.O. (HL-58655), the Alabama Chapter of the American Lung Association
(to W.L.S.), and the National Institutes of Health to J.S.H. (HL-03239
and HD-28831).
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Mitchell A. Olman, MA, MD,
Department of Medicine, Division of Pulmonary and Critical Care
Medicine, University of Alabama at Birmingham Medical Center, 1900 University Blvd, 215 THT, Birmingham, AL 35294; e-mail:
Olman{at}pulm.dom.uab.edu.
| |
REFERENCES |
1.
Dvorak HF, Senger DR, Dvorak AM, Harvey VS, McDonagh J:
Regulation of extravascular coagulation by microvascular permeability.
Science
227:1059, 1985[Abstract/Free Full Text]
2.
Dvorak HF, Harvey VS, Estrella P, Brown LF, McDonagh J, Dvorak AM:
Fibrin containing gels induce angiogenesis. Implications for tumor stroma generation and wound healing.
Lab Invest
57:673, 1987[Medline]
[Order article via Infotrieve]
3.
Brown LF, Dvorak AM, Dvorak HF:
Leaky vessels, fibrin deposition, and fibrosis: a sequence of events common to solid tumors and to many other types of disease [review].
Am Rev Resp Dis
140:1104, 1989[Medline]
[Order article via Infotrieve]
4.
Olman M, Mackman N, Gladson C, Moser K, Loskutoff D:
Changes in procoagulant and fibrinolytic gene expression during bleomycin induced lung injury in the mouse.
J Clin Invest
96:1621, 1995
5.
Bugge TH, Kombrinck KW, Flick MJ, Daugherty CC, Danton MS, Degen JL:
Loss of fibrinogen rescues mice from the pleiotropic effects of plasminogen deficiency.
Cell
87:709, 1996[Medline]
[Order article via Infotrieve]
6.
Smith EB, Thompson WD:
Fibrin as a factor in atherogenesis.
Thromb Res
73:1, 1995
7.
Ny T, Sawdey M, Lawrence DA, Millan JL, Loskutoff DJ:
Cloning and sequence of a cDNA coding for the human -migrating endothelial-cell-type plasminogen activator inhibitor.
Proc Natl Acad Sci USA
83:6776, 1986[Abstract/Free Full Text]
8.
van Mourik JA, Lawrence DA, Loskutoff DJ:
Purification of an inhibitor of plasminogen activators (antiactivator) synthesized by endothelial cells.
J Biol Chem
259:14914, 1984[Abstract/Free Full Text]
9.
Loskutoff DJ, Sawdey M, Mimuro J:
Type 1 plasminogen activator inhibitor, in
Coller B
(ed):
Progress in Hemostasis and Thrombosis. Columbia, MD, Bermedica Production, 1989, p 87.
10.
Loskutoff DJ, Schleef RR:
Plasminogen activators and their inhibitors.
Methods Enzymol
163:293, 1988[Medline]
[Order article via Infotrieve]
11.
Flaumenhaft R, Abe M, Mignatti P, Rifkin DB:
Basic fibroblast growth factor-induced activation of latent transforming growth factor in endothelial cells: Regulation of plasminogen activator activity.
J Cell Physiol
118:901, 1992
12.
Kirchheimer JC, Binder BR, Remold HG:
Matrix-bound plasminogen activator inhibitor type 1 inhibits the invasion of human monocytes into interstitial tissue.
J Immunol
145:1518, 1990[Abstract]
13.
Deng G, Curriden SA, Wang S, Rosenberg S, Loskutoff DJ:
Is plasminogen activator inhibitor-1 the molecular switch that governs urokinase receptor-mediated cell adhesion and release.
J Cell Biol
134:1563, 1996[Abstract/Free Full Text]
14.
Waltz DA, Natkin L, Fujita RM, Wei Y, Chapman HA:
Plasmin and plasminogen activator inhibitor type 1 promote cellular motility by regulating the interaction between the urokinase receptor and vitronectin.
J Clin Invest
100:58, 1997[Medline]
[Order article via Infotrieve]
15.
Stefansson S, Lawrence DA:
The serpin PAI-1 inhibits cell migration by blocking intergrin V3 binding to vitronectin.
Nature
383:441, 1996[Medline]
[Order article via Infotrieve]
16.
Carmeliet P, Stassen JM, Schoonjans L, Ream B:
Plasminogen activator inhibitor-1 gene-deficient mice. II. Effects on hemostasis, thrombosis, and thrombolysis.
J Clin Invest
92:2756, 1993
17.
Eitzman DT, McCoy RD, Zheng X, Fay WP, Shen T, Ginsburg D, Simon RH:
Bleomycin-induced pulmonary fibrosis in transgenic mice that either lack or overexpress the murine plasminogen activator inhibitor-1 gene.
J Clin Invest
1:232, 1996
18.
Greiling D, Clark RAF:
Fibronectin provides a conduit for fibroblast transmigration from collagenous stroma into fibrin clot provisional matrix.
J Cell Sci
110:861, 1997[Abstract]
19.
Seebacher T, Manske M, Zoller J, Crab J, Bade EG:
The EGF-inducible protein EIP-1 of migrating normal and malignant rat liver epithelial cells in identical to plasminogen activator inhibitor 1 and is a component of the ECM migration tracks.
Exp Cell Res
203:504, 1992[Medline]
[Order article via Infotrieve]
20.
Crutchley DJ, Conanan LB, Maynard JR:
Human fibroblasts produce inhibitor directed against plasminogen activator when treated with glucocorticoids.
Ann NY Acad Sci
370:609, 1981[Medline]
[Order article via Infotrieve]
21.
Laiho M, Saksela O, Andreasen PA, Keski-Oja J:
Enhanced production and extracellular deposition of the endothelial-type plasminogen activator inhibitor in cultured human lung fibroblasts by transforming growth factor-.
J Cell Physiol
103:2403, 1986
22.
Lund LR, Riccio A, Andreasen PA, Nielsen LS, Kristensen P, Laiho M, Saksela O, Blasi F, Dano K:
Transforming growth factor- is a strong and fast acting positive regulator of the level of type-1 plasminogen activator inhibitor mRNA in WI-38 human lung fibroblasts.
EMBO J
6:1281, 1987[Medline]
[Order article via Infotrieve]
23.
Circolo A, Welgus HG, Pierce GF, Kramer J, Strunk RC:
Differential regulation of the expression of proteinases/antiproteinases in fibroblasts.
J Biol Chem
266:12283, 1991[Abstract/Free Full Text]
24.
Idell S, Zwieb C, Boggaram J, Holiday D, Johnson AR:
Mechanisms of fibrin formation and lysis by human lung fibroblasts influence of TGF-beta and TNF-alpha.
Am J Physiol
263:L487, 1992[Abstract/Free Full Text]
25.
Samad F, Bergtrom G, Amrani DL:
Regulation of plasminogen activation by interleukin-6 in human lung fibroblasts.
Biochimica Biophys Acta
1221:307, 1994[Medline]
[Order article via Infotrieve]
26.
Olman MA, Simmons WL, Pollman DJ, Loftis AY, Bini A, Miller EJ, Fuller GM, Rivera K:
Polymerization of fibrinogen in murine bleomycin-induced lung injury.
Am J Physiol
271:L519, 1996[Abstract/Free Full Text]
27.
Hagood JS, Olman MA, Zeballos JG, Rivera KE, Fuller GM:
Regulation of type I plasminogen activator inhibitor by fibrin degradation products in rat lung fibroblasts.
Blood
87:3749, 1996[Abstract/Free Full Text]
28.
Bruzdzinski CJ, Riordan-Johnson M, Nordby EC, Suter SM, Gelehrter TD:
Isolation and characterization of the rat plasminogen activator inhibitor-1 gene.
J Biol Chem
265:2078, 1990[Abstract/Free Full Text]
29.
Johnson MR, Bruzdzinski CJ, Winograd SS, Gelehrter TD:
Regulatory sequences and protein-binding sites involved in the expression of the rat plasminogen activator inhibitor-1 gene.
J Biol Chem
267:12202, 1992[Abstract/Free Full Text]
30.
Olive M, Krylov D, Echlin DR, Gardner K, Taparowsky E, Vinson C:
A dominant negative to activation protein-1 (AP1) that abolishes DNA binding and inhibits oncogenesis.
J Biol Chem
272:18586, 1997[Abstract/Free Full Text]
31.
Olman MA, Williams WF, Strickland JH, Hagood JS, Simmons WL, Rivera KE:
Facile purification of fibrin(ogen) fragments using a computer based model with general applicability to the generation of salt gradients.
Prot Exp Purif
14:71, 1998[Medline]
[Order article via Infotrieve]
32.
Gailit J, Clarke C, Newman D, Tonnesen MG, Mosesson MW, Clark RA:
Human fibroblasts bind directly to fibrinogen at RGD sites through integrin (v)3.
Exp Cell Res
232:118, 1997[Medline]
[Order article via Infotrieve]
33.
Pizzo SV, Taylor LM Jr, Schwartz ML, Hill RL, McKee PA:
Subunit structure of fragment D from fibrinogen and cross-linked fibrin.
J Biol Chem
248:4584, 1973[Abstract/Free Full Text]
34.
Francis CW, Marder VJ, Barlow GH:
Plasmic degradation of crosslinked fibrin.
J Clin Invest
66:1033, 1980
35.
Zhang Z, Fuentes NL, Fuller GM:
Characterization of the IL-6 responsive elements in the gamma fibrinogen gene promoter.
J Biol Chem
270:24287, 1995[Abstract/Free Full Text]
36.
Liu A, Fuller GM:
Detection of a novel transcription factor for the A fibrinogen gene in response to interleukin-6.
J Biol Chem
270:7580, 1995[Abstract/Free Full Text]
37.
Zar JH:
Biostatistical Analysis. Englewood Cliffs, NJ, Prentice-Hall, 1984.
38.
Keeton MR, Curriden SA, van Zonneveld A, Loskutoff D:
Identification of regulatory sequences in the type 1 plasminogen activator inhibitor gene responsive to transforming growth factor .
J Biol Chem
266:23048, 1991[Abstract/Free Full Text]
39.
Knudsen H, Olesen T, Riccio A, Ungaro P, Christensen L, Andreasen PA:
A common response element mediates differential effects of phorbol esters and forskolin on type-1 plasminogen activator inhibitor gene expression in human breast carcinoma cells.
Eur J Biochem
220:63, 1994[Medline]
[Order article via Infotrieve]
40.
Descheemaeker KA, Wyns S, Nelles L, Auwerx J, Ny T, Collen D:
Interaction of AP-1-, AP-2, and Sp1-like proteins with two distinct sites in the upstream regulatory region of the plasminogen activator inhibitor-1 gene mediates the phorbol 12-myristate 13-acetate response.
J Biol Chem
267:15086, 1992[Abstract/Free Full Text]
41.
Ryan MP, Higgins PJ:
Growth state-regulation expression of p52(PAI-1) in normal rat kidney cells.
J Cell Physiol
155:376, 1993[Medline]
[Order article via Infotrieve]
42.
Angel P, Karin M:
The role of Jun, Fos and the AP-1 complex in cell-proliferation and transformation [review].
Biochim Biophys Acta
1072:129, 1991[Medline]
[Order article via Infotrieve]
43.
Lau LF, Nathans D:
Expression of a set of growth-related immediate early genes in BALB/c 3T3 cells: Coordinate regulation with c-fos or c-myc.
Proc Natl Acad Sci USA
84:1182, 1987[Abstract/Free Full Text]
44.
Brown JR, Nigh E, Lee RJ, Ye H, Thompson MA, Saudo F, Pestell RG, Greenberg ME:
Fos family members induce cell cycle entry by activating cyclin D1.
Mol Cell Biol
18:5609, 1998[Abstract/Free Full Text]
45.
Kovary K, Bravo R:
The Jun and Fos protein families are both required for cell cycle progression in fibroblasts.
Mol Cell Biol
11:4466, 1991[Abstract/Free Full Text]
46.
Johnson RS, Van Lingen B, Papaioannou VE, Spiegelman BM:
A null mutation at the c-jun locus causes embryonic lethality and retarded cell growth in culture.
Genes Dev
7:1309, 1993[Abstract/Free Full Text]
47.
Deng G, Curriden SA, Wang S, Rosenberg S, Loskutoff DJ:
Is plasminogen activator inhibitor-1 the molecular switch that governs urokinase receptor-mediated cell adhesion and release.
J Cell Biol
134:1563, 1996
48.
Odrljin TM, Shainoff JR, Lawrence SO, Simpson-Haidaris PJ:
Thrombin cleavage enhances exposure of a heparin binding domain in the N-terminus of the fibrin chain.
Blood
88:2050, 1996[Abstract/Free Full Text]
49.
Erban JK, Wagner DD:
A 130-kDa protein on endothelial cells binds to amino acids 15-42 of the B chain of fibrinogen.
J Biol Chem
267:2451, 1992[Abstract/Free Full Text]
50.
Bini A, Kudryk BJ:
Fibrinogen and fibrin in the arterial wall [review].
Thromb Res
75:337, 1994[Medline]
[Order article via Infotrieve]
51.
Bini A, Fenoglio J, Sobel J, Owen J, Fejgl M, Kaplan KL:
Immunochemical characterization of fibrinogen, fibrin I, and fibrin II in human thrombi and atherosclerotic lesions.
Blood
69:1038, 1987[Abstract/Free Full Text]
52.
Davis RJ:
The mitogen-activated protein kinase signal transduction pathway.
J Biol Chem
268:14553, 1993[Free Full Text]
53.
Claret FX, Hibi M, Dhut S, Toda T, Karin M:
A new group conserved coactivators that increase the specificity of AP-1 transcription factors.
Nature
383:453, 1996[Medline]
[Order article via Infotrieve]
54.
Karin M:
The regulation of AP-1 activity by mitogen-activated protein kinases [review].
J Biol Chem
270:16483, 1995[Free Full Text]
55.
Romer J, Bugge TH, Pyke C, Lund LR, Flick MJ, Degen JL, Dano K:
Imparied wound healing in mice with a disrupted plasminogen gene.
Nat Med
2:287, 1996[Medline]
[Order article via Infotrieve]
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
U. R. Jag, J. Zavadil, and F. M. Stanley
Insulin Acts through FOXO3a to Activate Transcription of Plasminogen Activator Inhibitor Type 1
Mol. Endocrinol.,
October 1, 2009;
23(10):
1587 - 1602.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Q. Ding, C. L. Gladson, H. Wu, H. Hayasaka, and M. A. Olman
Focal Adhesion Kinase (FAK)-related Non-kinase Inhibits Myofibroblast Differentiation through Differential MAPK Activation in a FAK-dependent Manner
J. Biol. Chem.,
October 3, 2008;
283(40):
26839 - 26849.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. K. Vayalil, M. Olman, J. E. Murphy-Ullrich, E. M. Postlethwait, and R.-M. Liu
Glutathione restores collagen degradation in TGF-{beta}-treated fibroblasts by blocking plasminogen activator inhibitor-1 expression and activating plasminogen
Am J Physiol Lung Cell Mol Physiol,
December 1, 2005;
289(6):
L937 - L945.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. Hilfiker-Kleiner, A. Hilfiker, K. Kaminski, A. Schaefer, J.-K. Park, K. Michel, A. Quint, M. Yaniv, J. B. Weitzman, and H. Drexler
Lack of JunD Promotes Pressure Overload-Induced Apoptosis, Hypertrophic Growth, and Angiogenesis in the Heart
Circulation,
September 6, 2005;
112(10):
1470 - 1477.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. Hageman, B. J. Eggen, T. Rozema, K. Damman, H. H. Kampinga, and R. P. Coppes
Radiation and Transforming Growth Factor-{beta} Cooperate in Transcriptional Activation of the Profibrotic Plasminogen Activator Inhibitor-1 Gene
Clin. Cancer Res.,
August 15, 2005;
11(16):
5956 - 5964.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. Prabhakaran, L. B. Ware, K. E. White, M. T. Cross, M. A. Matthay, and M. A. Olman
Elevated levels of plasminogen activator inhibitor-1 in pulmonary edema fluid are associated with mortality in acute lung injury
Am J Physiol Lung Cell Mol Physiol,
July 1, 2003;
285(1):
L20 - L28.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T.-L. Tuan, H. Wu, E. Y. Huang, S. S. N. Chong, W. Laug, D. Messadi, P. Kelly, and A. Le
Increased Plasminogen Activator Inhibitor-1 in Keloid Fibroblasts May Account for their Elevated Collagen Accumulation in Fibrin Gel Cultures
Am. J. Pathol.,
May 1, 2003;
162(5):
1579 - 1589.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. A. Eddy
Plasminogen activator inhibitor-1 and the kidney
Am J Physiol Renal Physiol,
August 1, 2002;
283(2):
F209 - F220.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. I. Vulin and F. M. Stanley
A Forkhead/Winged Helix-related Transcription Factor Mediates Insulin-increased Plasminogen Activator Inhibitor-1 Gene Transcription
J. Biol. Chem.,
May 31, 2002;
277(23):
20169 - 20176.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
N. Wang, L. Verna, H.-l. Liao, A. Ballard, Y. Zhu, and M. B. Stemerman
Adenovirus-Mediated Overexpression of Dominant-Negative Mutant of c-Jun Prevents Intercellular Adhesion Molecule-1 Induction by LDL: A Critical Role for Activator Protein-1 in Endothelial Activation
Arterioscler Thromb Vasc Biol,
September 1, 2001;
21(9):
1414 - 1420.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. S. Andrew, L. R. Klei, and A. Barchowsky
AP-1-dependent induction of plasminogen activator inhibitor-1 by nickel does not require reactive oxygen
Am J Physiol Lung Cell Mol Physiol,
September 1, 2001;
281(3):
L616 - L623.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. P. INGENITO, J. J. REILLY, S. J. MENTZER, S. J. SWANSON, R. VIN, H. KEUHN, R. L. BERGER, and A. HOFFMAN
Bronchoscopic Volume Reduction . A Safe and Effective Alternative to Surgical Therapy for Emphysema
Am. J. Respir. Crit. Care Med.,
July 15, 2001;
164(2):
295 - 301.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K.-S. Choi, S. L. Fitzpatrick, N. R. Filipenko, D. K. Fogg, G. Kassam, A. M. Magliocco, and D. M. Waisman
Regulation of Plasmin-dependent Fibrin Clot Lysis by Annexin II Heterotetramer
J. Biol. Chem.,
June 29, 2001;
276(27):
25212 - 25221.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
TOP
ABSTRACT
INTRODUCTION