|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 12 (December 15), 1999:
pp. 4177-4185
Induction of the Plasminogen Activator Inhibitor-1 Gene Expression by
Mild Hypoxia Via a Hypoxia Response Element Binding the
Hypoxia-Inducible Factor-1 in Rat Hepatocytes
By
Thomas Kietzmann,
Ulrike Roth, and
Kurt Jungermann
From the Institut für Biochemie und Molekulare Zellbiologie,
Göttingen, Germany.
 |
ABSTRACT |
Plasminogen activator inhibitor-1 (PAI-1) is the primary
physiological inhibitor of both tissue-type and urokinase-type
plasminogen activators. The balance between plasminogen activators and
PAI-1 plays an important role in several physiological and
pathophysiological processes such as atherosclerosis or thrombosis.
Because these conditions are associated with hypoxia, it was the aim of
the present study to investigate the influence of low O2
tension on the expression of PAI-1 mRNA and protein using primary
cultured rat hepatocytes as a model system. We found that PAI-1 mRNA
and protein were induced by mild hypoxia (8% O2). The
hypoxia-dependent PAI-1 mRNA induction was transcriptionally regulated
because it was inhibited by actinomycin D (ActD). Luciferase (LUC)
reporter gene constructs driven by about 800 bp of the
5'-flanking region of the rat PAI-1 gene were transiently
transfected into primary rat hepatocytes; mild hypoxia caused a 3-fold
induction, which was mediated by the PAI-1 promoter region -175/-158
containing 2 putative hypoxia response elements (HRE) binding the
hypoxia-inducible factor (HIF-1). Mutation of the HRE-1 (-175/-168) or
HRE-2 (-165/-158) also abolished the induction by mild hypoxia.
Cotransfection of a HIF-1 vector and the PAI-1-LUC constructs, as
well as gel shift assays, showed that the HRE-2 of the PAI-1 promoter
was most critical for induction by hypoxia and HIF-1 binding. Thus,
PAI-1 induction by mild hypoxia via a HIF-1 binding HRE in the rat
PAI-1 promoter appears to be the mechanism causing the increase in
PAI-1 in many clinical conditions associated with O2 deficiency.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
PLASMINOGEN ACTIVATOR inhibitors (PAIs)
play a crucial role in physiological and pathophysiological processes,
which involve the tissue-type plasminogen-activator (tPA) and the
urokinase (uPA) catalyzed conversion of plasminogen to
plasmin.1,2 Two types of PAIs named PAI-13 and
PAI-24 could be identified on the basis of biochemical,
immunologic, and molecular biological properties. Whereas the
physiological function of PAI-2 has not been clearly
elucidated,5 PAI-1 is the major plasminogen activator
inhibitor.6 PAI-1 can be produced and secreted by
platelets, vascular endothelial cells,7 vascular smooth
muscle cells,8 and several nonvascular cell
types,9,10 among them hepatocytes.11
Furthermore, PAI-1 is found also in the extracellular
matrix.12 Thus, PAI-1 appears to be a key inhibitor of
fibrinolysis3,13 and of proteolytic processes, which are
associated with neovascularization, tissue remodelling, and
regeneration, such as inflammation14 and
fibrosis.15
The importance of the balance between plasminogen activators and PAI-1
has been emphasized by several clinical studies: decreased PAI-1 levels
are associated with bleeding diathesis,16-18 whereas increased PAI-1 levels have been found in a number of clinical conditions, such as sepsis,19 atherosclerotic lesions
leading to myocardial infarction,20 and deep vein
thrombosis.21 In a rabbit model of thrombosis, increased
levels of PAI-1 promoted thrombus extension,22 and
inhibition of PAI-1 activity by a monoclonal antibody against PAI-1
partially prevented thrombus formation.23 Moreover, in
rats, hemorrhage increased the expression of PAI-1 in various tissues,
such as lung, kidney, and liver.24 Thus, PAI-1 may promote
thrombus extension and the development of thromboembolic diseases.
Because prethrombotic events, hemorrhage, and thrombus formation are
associated with hypoxia, it was the goal of this study to investigate
the influence of reduced O2 tensions on the expression of
PAI-1. Primary cultured rat hepatocytes were used as a model system. We
found that PAI-1 mRNA and protein were induced by mild hypoxia (8%
O2). The enhancement of PAI-1 gene expression by mild hypoxia was mainly regulated on the level of transcription via an
O2 responsive sequence (-175/-158) containing the potential hypoxia responsive elements -1 and -2 (HRE-1, -175/-168 and HRE-2, -165/-158) binding the hypoxia-inducible factor-1 (HIF-1). Transfection of PAI-1 promoter luciferase (LUC) gene constructs and gel shift assays
showed that the HRE-2 had a key role in PAI-1 gene activation by
hypoxia and HIF-1 binding. Thus, HIF-1 appears to be most critical for
the increase in PAI-1 under hypoxia.
| |
MATERIALS AND METHODS |
All biochemicals and enzymes were of analytical grade and were
purchased from commercial suppliers.
Animals.
Male Wistar rats (200 to 260 g) were kept on a 12-hour day/night rhythm
(light from 7 AM to 7 PM) with free access to water and food. Rats were anesthetized with pentobarbital (60 mg/kg body
weight) before preparation of hepatocytes between 8 AM and 9 AM.
Cell culture experiments.
Liver cells were isolated by collagenase perfusion. Cells (1 × 106 per dish) were maintained under standard conditions in
a normoxic atmosphere of 16% O2, 79% N2, and
5% CO2 (by vol) in medium M199 containing 0.5 nmol/L
insulin added as a growth factor for culture maintenance, 100 nmol/L
dexamethasone required as a permissive hormone, and 4% fetal calf
serum for the initial 4 hours of culture. Cells were then cultured in
serum-free medium from 4 to 24 hours at normal arterial 16%
O2. At 24 hours, medium was changed and culture was
continued at normoxia 16% O2 or at mild hypoxia 8% O2 (87% N2, 5% CO2 [by vol]).
Sixteen percent O2 and 8% O2 at the medium
surface correspond to 13% O2 = arterial pO2
and 6% O2 = venous pO2 at the cell
surface.25 In actinomycin and cycloheximide experiments,
hepatocytes were pretreated for 30 minutes with ActD (1 µg/mL) or
cycloheximide (10 µg/mL) before the cells were cultured under
normoxia or mild hypoxia for 3 hours.
RNA preparation and Northern analysis.
Isolation of total RNA and Northern analysis were performed as
described.26 Digoxigenin (DIG)-labelled antisense RNAs
served as hybridization probes; they were generated by in vitro
transcription from pBS-PAI-1 using T3 RNA polymerase and RNA labeling
mixture containing 3.5 mmol/L 11-DIG-uridine triphosphate
(UTP), 6.5 mmol/L UTP, 10 mmol/L guanosine triphosphate
(GTP), 10 mmol/L cytidine triphosphate (CTP),
10 mmol/L adenosine triphosphate (ATP). Hybridizations were performed with 50 ng/mL transcript at 68°C for 6 hours
according to the manufacturer's application notes of the DIG-nucleic
acid detection kit (Roche, Mannheim, Mannheim, Germany).
Detection of hybrids was performed as described
previously.26 Blots were quantified with a
videodensitometer (Biotech Fischer, Reiskirchen, Germany).
Plasmid constructs.
The rat PAI-1 promoter 5'-flanking region27 from -766 to +31 was amplified by polymerase chain reaction (PCR) from rat
genomic DNA by using the oligonuclotide
5'-gacagagctctctgtggtaacccctgtgctc-3' (-766/-736) as
forward and 5'-tcggagatctgcagcagcctgatccagctgtg-3' (-1/+31)
as reverse primer, respectively. The resulting PCR product was digested
with SacI and BglII and ligated into the SacI
and BglII sites of pGl3basic (Promega, Mannheim,
Germany) (pGl3PAI-766, see Fig 3).
In construct pGl3PAI-766 M, the -175/-159 fragment was omitted by
deletion mutagenesis with the ExSite Mutagenesis kit (Stratagene, La
Jolla, CA) by using the pGl3PAI-766 as template and the
oligonucleotides 5'-tcccagcaagttactgggaggga-3' (-158/-136)
as forward primer and 5'-gtgagagggcatgggggataattg-3'
(-176/-199) as reverse primer, respectively. The constructs
pGl3PAI-766M1 (see Fig 3) and pGl3PAI-766M2 (see Fig 3) were
constructed by the Altered Sites in vitro Mutagenesis kit (Promega)
using the mutagenized oligonucleotides M1 and M2 shown in
Table 1.
The plasmid pcDNAHIF-1 containing the full-length rat HIF-1 cDNA
under the control of the cytomegalovirus (CMV) promoter was constructed
by excision of the HIF-1 cDNA (Gene bank Y09507) from pCRII with
BamHI and EcoRI and subsequent ligation into the BamHI, EcoRI sites of pcDNAI-Amp (Invitrogen,
Groningen, The Netherlands). All constructs were verified
by sequencing in both directions.
Cell transfection and LUC assay.
Freshly isolated rat hepatocytes (about 1 × 106 cells
per dish) were transfected for 5 hours with 2.5 µg plasmid DNA
containing 500 ng of a Renilla luciferase (RL) reporter plasmid
(pRL-SV40, Promega) to control transfection efficiency and 2 µg of
the appropriate PAI-1 promoter Firefly luciferase (FL)
construct essentially as described.28 In HIF-1
cotransfection assays, 2 µg of the appropriate PAI-1 promoter FL
construct were transfected together with 200 ng pcDNAHIF or in the
controls with pcDNA, thereby reducing the amount of pRL-SV40 to 300 ng.
After 5 hours, the medium was changed and the cells were cultured under
normoxia for 19 hours. The medium was changed again and the cells were
further cultured for 24 hours under normoxia or mild hypoxia.
Western blot analysis.
Western blot analysis was performed as described.29 In
brief, media from primary cultured hepatocytes were collected and the
protein content was determined using the Bradford method. A total of 50 µg of protein was loaded onto a 10% sodium dodecyl sulfate
(SDS)-polyacrylamide gel and after electrophoresis blotted onto
nitrocellulose membranes. The primary rabbit antibody against rat PAI-1
(American Diagnostics, Pfungstadt, Germany) was used in a
1:200 dilution. The primary rabbit aryl hydrocarbon nuclear translocator (ARNT) antibody (Affinity Bioreagents, Grünwald, Germany) was used in a 1:500 dilution. The secondary
antibody was a goat antirabbit IgG (Santa Cruz Biotech,
Santa Cruz, CA) and used in a 1:2000 dilution. The ECL Western blotting
system (Amersham, Arlington Heights, IL) was then used
for detection. Under these conditions, PAI-1 was seen as a double band,
the major 49 kD band and the minor 46 kD band.29
Preparation of nuclear extracts.
Nuclear extracts were prepared by modification of a standard
protocol30,31 with buffers A and C containing 0.5 mmol/L
dithioerythritol (DTE; Sigma, St Louis, MO), 0.4 mmol/L
phenylmethylsulfonyl fluoride (Serva, Heidelberg,
Germany), 2 µg of leupeptin per mL (Boehringer), 2 µg
of pepstatin per mL (Boehringer), 2 µg of aprotinin per mL (Bayer, Leverkusen, Germany), 1 mmol/L sodium vanadate
(Sigma), and the "complete" protease inhibitor cocktail tablets
(Boehringer). Briefly, confluent HepG2 cells in 10-cm dishes were
cultured at 8% O2 or 16% O2 for 24 hours, the
cells were then washed twice with ice cold 0.9% NaCl, scraped into 300 µL NaCl, and pelleted by centrifugation at 2000 rpm for 2 minutes at
4°C in an Sorvall high-speed centrifuge RC 5B (SS 34 rotor). The cell pellet was resuspended in 4 packed cell
volumes (PCV) of buffer A (10 mmol/L Hepes-KOH [pH 7.9], 1.5 mmol/L
MgCl2, 10 mmol/L KCl), and incubated on ice for 10 minutes.
The cell suspension was Dounce homogenized with a type-B pestle, and
the nuclei were pelleted by centrifugation at 2300 rpm for 10 minutes,
resuspended in 3 PCV of buffer A, and pelleted once again by
centrifugation at 14,500 rpm for 20 minutes. The nuclei were
resuspended in 3 PCV of buffer C (20 mmol/L HEPES-KOH [pH 7.9], 1.5 mmol/L MgCl2, 0.42 mmol/L NaCl, 0.2 mmol/L EDTA, 20%
glycerol), and incubated at 4°C with gentle agitation for 30 minutes. Nuclear debris was pelleted by centrifugation for 30 minutes
at 14,500 rpm. The supernatant was dialyzed against a 50-fold volume of
buffer D (20 mmol/L Hepes-KOH [pH 7.9], 0.1 mmol/L KCl, 0.2 mmol/L
EDTA, 20% glycerol) for 4 hours. The dialysate was centrifuged for 10 minutes at 14,500 rpm, and aliquots of the supernatant were frozen in
liquid N2 and stored at  70°C. Protein
concentration was determined by the Bradford method with bovine serum
albumin standards.
Electrophoretic mobility shift assay (EMSA).
The sequences of the PAI-1 oligonucleotides used for the EMSA are shown
(see Fig 4). Equal amounts of complementary
oligonucleotides were annealed and labeled by 5'-end-labelling
with [ -32P]ATP (Amersham) and T4 polynucleotide kinase
(MBI). They were purified with the Nucleotide Removal Kit
(Quiagen, Hilden, Germany). Binding reactions were
performed in a total volume of 20 µL containing 250 mmol/L KCl, 5 mmol/L MgCl2, 5.5 mmol/L EDTA, 25% glycerol, 10 µg of
nuclear extract, 250 ng poly d(I-C), and 5 mmol/L DTE. After
preincubation for 5 minutes at room temperature, 1 µL of the labeled
probe (104 cpm) was added and the incubation was continued
for an additional 10 minutes. For supershift analysis, 1 µL preimmune
serum or a HIF-1 antibody (1 µL or 0.1 µL of a 1:5 dilution) was
added to the EMSA reaction and then incubated at 4°C for 2 hours.
The antibodies against rat-HIF-1 were raised in rabbits against a
223 amino acid peptide (rat HIF aa 166-388).
The electrophoresis was then performed with a 5% nondenaturing
polyacrylamide gel in TBE buffer (89 mmol/L Tris, 89 mmol/L boric acid,
5 mmol/L EDTA) at 200 V. After electrophoresis, the gels were dried and
exposed to a phosphorimager screen.
| |
RESULTS |
Induction of PAI-1 mRNA and protein by mild hypoxia.
In primary cultured rat hepatocytes, mild hypoxia (8% O2)
induced PAI-1 mRNA within 2 hours by about 2.5-fold
(Fig 1) and within 8 hours by about
3.5-fold (Fig 1). Induced PAI-1 mRNA then remained almost stable at
this level up to 24 hours (data not shown). The increase of PAI-1 mRNA
by mild hypoxia was followed by an increase of PAI-1 protein. PAI-1
protein was first detectable in the medium of the cells after 8 hours;
under mild hypoxia, the amount of PAI-1 protein was increased by about
2-fold compared with normoxia (Fig 1). After 24 hours exposure to mild
hypoxia, the amount of PAI-1 protein was further increased by about
3-fold (Fig 1). Thus, PAI-1 mRNA and protein expression in primary rat hepatocytes were induced by mild hypoxia.
View larger version (32K):
[in this window]
[in a new window]
| Fig 1.
Hypoxia-dependent induction of PAI-1 mRNA and protein
expression in primary rat hepatocyte cultures. Hepatocytes were
cultured for 24 hours under arterial pO2. At 24 hours, the
medium was changed and cells were further cultured for the times
indicated under normoxic (16% O2) and hypoxic (8%
O2) conditions. (A) In each experiment, the PAI-1 mRNA
level measured by Northern blotting (cf B) at 0 hour
under normoxia (16% O2) was set equal to 100%. The PAI-1
protein level measured by Western blotting (cf B) at 0 hour under
normoxia (16% O2) was set equal to 1%. Values are means ± standard error of mean (SEM) of 3 independent culture
experiments. Statistics, Student's t-test for paired values: *
significant differences 16% O2 versus 8% O2,
P  .05. (B) Representative Northern and Western blot. For
Northern analysis, 15 µg total RNA prepared from the cultured
hepatocytes were hybridized to DIG-labelled PAI-1 and  -actin
antisense RNA probes (cf, Materials and Methods). A total of 50 µg of
protein from the medium of the cultured hepatocytes was subjected to
Western analysis with an antibody against rat PAI-1 (cf, Materials and
Methods). Autoradiographic signals were obtained by chemiluminescence
and scanned by videodensitometry.
|
|
Transcriptional and translational regulation of the mild
hypoxia-dependent PAI-1 mRNA induction.
In the primary rat hepatocyte cultures, the transcriptional inhibitor
ActD prevented the mild hypoxia-dependent PAI-1 mRNA induction within 3 hours (Fig 2). Thus, PAI-1 gene activation by mild hypoxia occurred mainly on the transcriptional level. The
protein synthesis inhibitor cycloheximide caused an induction of PAI-1
mRNA under normoxia to levels normally induced by mild hypoxia, but it
did not increase any further the induction elicited by mild hypoxia
(Fig 2). The induction by cycloheximide under normoxia was probably due
to a stabilization of PAI-1 mRNA11; the lack of an
additivity of induction by cycloheximide and mild hypoxia may indicate
that the mild hypoxia-dependent induction of PAI-1 mRNA was dependent
on ongoing protein synthesis of, eg, HIF-1. Thus, the present finding
would be in line with previous reports showing that cycloheximide
(CHX) blocked the hypoxic induction of HIF-dependent
genes.31
View larger version (22K):
[in this window]
[in a new window]
| Fig 2.
Inhibition of the hypoxia-dependent PAI-1 mRNA induction
by ActD and cycloheximide in primary rat hepatocyte cultures.
Hepatocytes were cultured as in Fig 1. (A) The cells were then treated
for 30 minutes with ActD (1 µg/mL) or cycloheximide (CHX; 10 µg/mL)
before they were placed under hypoxia for 3 hours. A total of 15 µg
total RNA was subjected to Northern analysis. In each experiment, the
PAI-1 mRNA level measured by Northern blotting (cf B) under normoxia
(16% O2) was set equal to 100%. The induction of PAI-1
mRNA by cycloheximide under normoxia was due to a stabilization of
PAI-1 mRNA.11 Values ± SEM represent the fold induction
of PAI-1 mRNA of 3 independent experiments. Statistics, Student's
t-test for paired values: significant differences * 16%
O2 versus 8% O2, ** 8% O2 versus
ActD, P .05. (B) Representative Northern blot.
|
|
Identification of sequences responsible for the mild
hypoxia-dependent transcription from the PAI-1 promoter.
Sequence analysis of the rat PAI-1 promoter showed that 2 potential
hypoxia response elements (HRE), which might bind HIF-1, are present
within the first 200 bp of the promoter. Both are located inside the
"C"-site identified by footprint analysis with liver nuclear
extracts.27 The first potential HIF-1 binding element
(HRE-1) inside the "C-site" -175/-168 5'-CACGTACA-3'
matches the HIF-1 binding consensus sequence BACGTSSK32
(with B=G/C/T, S=G/C, and K=G/T) in 6 of 8 bp. The second potential
HIF-1 binding element (HRE-2) -165/-158 5'-CACGTGTC-3' also
matches the HIF-1 consensus site in 6 of 8 bp
(Fig 3A).
View larger version (25K):
[in this window]
[in a new window]
| Fig 3.
Regulation of transfected rat PAI-1 promoter LUC gene
constructs by hypoxia and cotransfected HIF-1 in transiently
transfected rat hepatocyte cultures. Enhancement of PAI-1 production by
HIF-1. (A) The 5'-flanking region of the rat PAI-1 gene with
its footprinted sites [A-G]. In the "C-site", the 2 potential
HIF-1 binding elements (HRE) matching the HIF-1 consensus sequence
BACGTSSK32 were B=G/C/T, S=G/C and K=G/T are
underlined. B, S, or K are shown only if the actual PAI-1 sequence does
not match any of the bases allowed by the consensus. (B) Hepatocytes
were transiently cotransfected with pcDNAHIF-1 containing the
full-length rat HIF-1 cDNA and LUC gene constructs driven by a
wild-type 766 bp rat PAI-1 promoter (pGl3PAI-766), or the 766 bp
promoter mutated at either the HRE-1 (pGl3PAI-766M1) or HRE-2
(pGl3PAI-766M2) site. After 24 hours, the transfected cells were
cultured for 18 hours under hypoxia, additionally
pGl3PAI-766-transfected cells were treated with CHX (10 µg/mL). In
each experiment, the fold stimulation of LUC activity by hypoxia (8%
O2) was determined relative to the normoxic (16%
O2) control, which was set equal to 1. The values represent
means ± SEM of 3 independent experiments. Statistics, Student's
t-test for paired values: * significant differences 16%
O2 versus 8% O2, P .05; **
significant differences 16% O2 versus 16% O2 + HIF-1, P .05. In pGl3PAI-766M1 and pGl3PAI-766M2,
the wild-type PAI-1 sequence is shown on the upper strand, mutated
bases are indicated by *, and are shown in lower case letters. (C)
Western blot analysis of PAI-1, HIF-1, and ARNT in cultured
hepatocytes transfected with a HIF-1 vector. A total of 50 µg of
protein from the medium and 100 µg of total cellular protein of the
transfected hepatocytes were subjected to Western analysis (cf,
Materials and Methods) with antibodies against rat PAI-1, rat HIF-1,
and ARNT, respectively. Blots were scanned by videodensitometry, and in
each experiment, the PAI-1 level and HIF-1 level measured under
normoxia (16% O2) were set equal to 1. Values represent
the fold induction of PAI-1 and HIF-1 of 3 independent
experiments.
|
|
A 766-bp fragment of the 5'-flanking region of rat PAI-1
gene27 was cloned in front of the LUC gene in pGl3-basic
(Promega) to generate pGl3PAI-766 (Fig 3). In hepatocytes transfected
with pGl3PAI-766, LUC activity was induced by about 3-fold under mild hypoxia (Fig 3B). To verify that the mild hypoxia-dependent induction of pGl3PAI-766 was dependent on ongoing protein synthesis of, eg,
HIF-1, pGl3PAI-766-transfected hepatocytes were treated with cycloheximide while cultured under mild hypoxia. It was found that
cycloheximide abolished the hypoxia-dependent induction of LUC activity
in transfected hepatocytes (Fig 3B). These results further support the
involvement of HIF-1 in the hypoxia-dependent PAI-1 gene activation.
Deletion of the "C"-site containing the 2 PAI-1-HREs in
pGl3PAI-766M abolished the hypoxia-dependent induction of LUC
activity in transfected hepatocytes indicating that these elements are necessary for the enhanced transcription of the PAI-1 gene under mild
hypoxia (data not shown). Mutation of the nucleotides -173/-167 encompassing the HRE-1 site in the construct pGl3PAI-766M1 resulted again in a loss of the enhancement of LUC activity by mild hypoxia in
transfected hepatocytes. When the nucleotides -165/-160 inside HRE-2
were mutated in the construct pGl3PAI-766M2, the mild hypoxia-elicited enhancement of LUC activity was also abolished (Fig 3B). These data
indicate that both HREs in the PAI-1 promoter contributed to the mild
hypoxia-dependent induction of PAI-1 gene transcription.
Activation of transfected rat PAI-1 promoter LUC gene constructs by
HIF-1.
To identify the direct regulation of the PAI-1 HREs by HIF-1, the
wild-type PAI-1 promoter LUC constructs and the mutated PAI-1 promoter
constructs were cotransfected into rat hepatocytes with an expression
vector encoding rat HIF-1. In hepatocytes transfected with
pGl3PAI-766 and the HIF-1 vector, the LUC activity was increased by
about 12-fold under 16% O2 (Fig 3B). Mild hypoxia and
cotransfected HIF-1 were synergistic in pGl3PAI-766-transfected hepatocytes; LUC activity was induced to about 18-fold (Fig 3B).
Cotransfection of pGl3PAI-766M1 mutated at HRE-1 and the HIF-1
vector resulted in an enhanced LUC activity by about 5-fold under
normoxia and hypoxia indicating that the remaining complete HRE-2 may
be sufficient for a HIF-1-dependent submaximal induction (Fig 3B).
In hepatocytes cotransfected with pGl3PAI-766M2 mutated in the HRE-2
and the HIF-1 vector, LUC activity under both O2
tensions was comparable to that obtained by transfection of the
wild-type PAI-1 promoter construct under normoxia and could not be
enhanced by mild hypoxia or HIF-1 (Fig 3B) despite the high presence
of HIF-1 in the cotransfected cultures (Fig 3C). This suggests that HRE-2 seems to be the main or even only HIF-1 target sequence in the
PAI-1 promoter.
Enhancement of PAI-1 protein production by transfection of rat
HIF-1.
Because the PAI-1 protein was also induced by mild hypoxia and this
response was mediated by HIF-1, it should be tested whether the PAI-1
production by rat hepatocytes could be increased by transfection of
HIF-1. We found that HIF-1-transfected hepatocytes secreted
about 2-fold more PAI-1 protein into the medium under 16%
O2 compared with the nontransfected control cells (Fig 3C). Again, mild hypoxia and transfected HIF-1 were synergistic; the amount of PAI-1 was increased by about 5-fold compared with the normoxic untransfected control and by about 2-fold compared with the
normoxic transfected control (Fig 3C). Thus, transfection of HIF-1
can mimic the response to mild hypoxia and enhance the production of
PAI-1 protein.
Binding of HIF-1 to sequences in the rat PAI-1 promoter.
The binding of nuclear proteins to oligonucleotide probes spanning the
putative HRE-1 and HRE-2 sites of the PAI-1 promoter was examined by EMSA.
The oligonucleotide -177/-152, which enclosed both the HRE-1 and HRE-2
site (Fig 4A), was able to bind, besides a
constitutive protein complex, a hypoxia-inducible nuclear protein
complex (Fig 4B, left panel). Thus, it seemed likely that the
hypoxia-induced DNA-protein complex contained HIF-1. To ensure this,
several dilutions of a specific antiserum against HIF-1 was included
in the binding reaction. The HIF-1 antibody was raised against a
peptide from amino acid 166 to 388 of the rat HIF-1 protein. This
region contains parts of the per-arnt-sim (PAS)-domain,
which is involved in protein dimerization. Thus, the addition of the
HIF-1 antibody should prevent the mild hypoxia-dependent DNA-protein
complex formation. With the -177/-152 PAI-1 (HRE-1/2) oligonucleotide,
this was indeed found (Fig 4B, left panel). Addition of 1 µL and 0.1 µL HIF-1 antibody to the EMSA reaction inhibited the formation of
the mild hypoxia-dependent DNA complex and led to a supershifted
complex, which could not be further resolved on the gel. Inhibition of the hypoxia-inducible complex was best seen with 1 µL of the HIF-1 antibody (Fig 4B, left panel). At this HIF-1 antibody concentration, binding of the so-called constitutive complex to the HRE1/2
oligonucleotide was en- hanced. With a further reduction of the
HIF-1 antibody (1:10), the formation of the hypoxia-inducible
complex returned (Fig 4B, left panel). Specificity of the HIF-1
antiserum was ensured by using the preimmune serum in the EMSA and in
the Western blot analysis (Fig 4C). When the preimmune serum was used,
the formation of the mild hypoxia-dependent DNA-protein complex was not
impaired (Fig 4B), and the presence of the HIF-1 protein in the
nuclear extracts could not be detected, whereas with the immune serum,
it was found that only under mild hypoxia the HIF-1 protein was
present in the nuclear extracts (Fig 4C).
View larger version (47K):
[in this window]
[in a new window]
| Fig 4.
Binding of HIF-1 to the rat PAI-1 promoter region. (A)
Oligonucleotides: the HIF-1 consensus sequence BACGTSSK32
were B=G/C/T, S=G/C, and K=G/T and the sense strand of the rat
PAI-1 promoter sequence -177/-152 containing the HRE-1 and HRE-2 site,
the sense strands of the oligonucleotides containing HRE-1, HRE-2, and
the oligonucleotide with a mutation in the HRE-2 site M-HRE are shown.
Bases matching the consensus sequences are underlined, mutated bases
are in lower case letters. (B) EMSA, left panel: the
32P-labeled PAI-1 HRE-1/2 oligonucleotide was incubated
with either 10 µg protein of nuclear extracts from normoxic (16%
O2) or hypoxic (8% O2) cells in the absence or
presence of preimmune serum or rat HIF-1 antibody as indicated (cf,
Materials and Methods). In EMSA with antibodies, the nuclear extracts
were preincubated with 1 µL of the HIF-1 antibody for 2 hours at
4°C before adding the labeled probe. Middle panel: the
32P-labeled HRE-2 (-168/-152) oligonucleotide and the PAI-1
HRE-1 (-182/-166) oligonucleotide were incubated with either 10 µg of
nuclear extracts from normoxic (16% O2) or hypoxic (8%
O2) cells in the absence or presence of preimmune serum or
rat HIF-1 antibody as indicated. The inducible (I) HIF-1 complex was
seen with the HRE-2, but not with the HRE-1 oligonucleotide. Right
panel: the 32P-labeled mutated HRE-2 (-168/-152)
oligonucleotide (M-HRE) was incubated with either 10 µg of nuclear
extracts from normoxic (16% O2) or hypoxic (8%
O2) cells or with extracts from hypoxic cells in the
presence of rat HIF-1 antibody as indicated. The image had to be
overexposed to visualize the weak constitutive binding. The DNA-protein
binding was analyzed by electrophoresis on 5% native polyacrylamide
gels. I, induced HIF-1 complex; C, constitutive complex; S,
supershifted complex. (C) Western analysis of nuclear extracts from
normoxic (16% O2) or hypoxic (8% O2) cells. A
total of 20 µg nuclear extract from 2 different preparations was
resolved on a 10% SDS gel and probed with preimmune serum or the
HIF-1 antibody.
|
|
When the oligonucleotide -168/-152 containing HRE-2 was used as a probe
in the EMSA, the HIF-1 complex was formed (Fig 4B, middle panel), but
the complex with the oligonucleotide containing HRE-1 and HRE-2
-177/-152 was stronger (Fig 4B, left panel). Again, the addition of 1 µL of the HIF-1 antibody prevented the HIF-1 complex formation,
enhanced formation of the constitutive complex, and formed the same
supershifted complex as with the HRE-1/2 probe (Fig 4B, middle panel).
The HIF-1 complex was also no longer detectabel when a mutation was
introduced in the HRE-2 site as with the M-HRE oligunucleotide (Fig 4B,
right panel). In contrast, with the PAI-1 promoter oligonucleotide
-182/-166 containing HRE-1, a very strong complex smaller than the
HIF-1 complex was detectable (Fig 4B, middle panel). The formation of
this DNA-protein complex was independent from the pO2, and
addition of the HIF-1 antibody did not interfere with the formation
of this strong complex. However, the HIF-1 antibody addition
resulted in the detection of a supershifted complex (Fig 4B, middle panel).
These data indicate that mainly the HRE-2 site in the PAI-1 promoter
was necessary for the interaction with HIF-1, and that the sequences in
the region -182/-166 had supporting functions in forming a stronger
HIF-1-DNA complex.
| |
DISCUSSION |
In this study, we show that PAI-1 mRNA and protein
expression were activated by hypoxia in primary rat hepatocytes (Figs 1 and 2), and that this induction was mediated by HIF-1 (Figs 3 and 4).
Transfection assays with rat PAI-1 promoter LUC gene constructs provided evidence that hypoxia activated PAI-1 via 2 HREs matching the
HIF-1 consensus sequence (Fig 3). Cotransfection of PAI-1 promoter LUC
gene constructs and full-length HIF-1 cDNA (Fig 3) and EMSAs (Fig 4)
showed that HIF-1 bound to HRE-2 and that HRE-1 had supportive
functions in the response of the rat PAI-1 promoter to hypoxia.
Functional and nonfunctional HIF-1 binding sites.
A variety of O2 responsive genes has been
identified.33 In the response to hypoxia, the transcription
factor HIF-1 appears to be a key regulator. HIF-1 is a heterodimer
consisting of HIF-1 and HIF-1, the latter being identical to the
ARNT protein.34 In the response to hypoxia, HIF-1 binds to
HREs, which can be located in the 5'-flanking or
3'-flanking regions of a gene. HIF-1 binding sites were
identified in a variety of hypoxia-inducible genes encoding
erythropoietin (EPO), vascular endothelial growth factor (VEGF), and
glycolytic enzymes.31,35 From these genes, the
HRE-consensus sequence 5'-BACGTSSK-3' (B=G/C/T, S=C/G,
K=G/T) was deduced.36 Furthermore, in a detailed analysis
of HREs in the human aldolase A (ALDA), enolase 1, and mouse lactate
dehydrogenase A gene promoters with transfection assays in Hep3B cells
and EMSAs, it was found that a HIF-1 sensitive DNA sequence should
consist of a pair of transcription factor binding sites, which
contained the HIF-1 core sequence 5'-RCGTG-3'
(R=A/G).37 Both the HRE-1 (-175/-168) and the HRE-2
(-165/-158) in the hypoxia-responsive DNA sequence of the rat PAI-1
promoter, -175/-157, identified in this study matched the HIF-1
consensus sequence 5'-BACGTSSK-3' in 6 of 8 bp (Fig 3A).
The HRE-2 contained the HIF-1 core sequence 5'-ACGTG-3',
whereas HRE-1 with 5'-ACGTA-3' did not. However, inspection
of the antisense strand showed that the antisense HRE-1 sequence
-170/-177 5'-TACGTGTG-3' and the antisense HRE-2 sequence -160/-167 5'-CACGTGTG-3' matches the HIF-1 binding
consensus sequence in 7 of 8 bp containing also the core sequence
5'-RCGTG-3'. This existence of 2 adjacent HIF-1 sites,
arranged as either direct or inverted repeats, has been noted in other
hypoxically regulated genes including human enolase 1,37
mouse lactate dehydrogenase A,37 human phosphoglycerate
kinase 1,38 and human transferrin.39
In hepatocytes transfected with either the PAI-1 promoter LUC
constructs pGl3PAI-766M1 or pGl3PAI-766M2, in which the HRE-1 and
HRE-2, respectively, were mutated (Fig 3B), the hypoxia-dependent increase in LUC activity was abolished (Fig 3B). Thus, both the HRE-1
and HRE-2 were involved in the hypoxia-dependent activation of the
PAI-1 promoter.
Cotransfection of hepatocytes with full-length HIF-1 cDNA and
pGl3PAI-766M2, in which the HRE-1, but not the HRE-2 was intact, did
not result in any induction of Luciferase (Fig 3). Gel shift analysis
with the 17-mer oligonucleotide -182/-166 spanning the HRE-1 did not
provide evidence for the same HIF-1 oligonucleotide complex as with the
HRE-1/2 or the HRE-2 probe (Fig 4). However, the presence of the
supershifted complex implicates that a small amount of HIF-1 was
present in this complex. This would mean that other partners of the
bHLH-protein family, such as USF or MYC, may interact with HIF-1 at
the HRE-1 of the PAI-1 promoter. Studies to clarify this question are
under way. However, these results clearly indicated that HRE-1 did not
bind HIF-1 with the same affinity as HRE-2 or HRE-1/2. In contrast,
cotransfection of hepatocytes with pGl3PAI-766M1 in which the HRE-2,
but not the HRE-1, was intact and a vector encoding full-length
HIF-1 cDNA caused induction of LUC activity (Fig 3). However, this
induction was not as strong as with the wild-type pGl3PAI-766 construct (Fig 3). The 17-mer oligonucleotide -168/-152 containing the HRE-2 formed the HIF-1 complex (Fig 4). These findings showed that HRE-2 bound HIF-1. Thus, it seems likely that HRE-1 is a low affinity or a
nonfunctional HIF-1 binding site. Such nonfunctional binding sites were
also identified in the human ALDA gene and in the mouse HIF-1 gene.
In the case with the ALDA gene, a part of the intervening sequence IV
(IVS-IV; intron D) 5'-CACGTGCG-3' from
+128/+13537 and in the promoter of HIF-1 exon
I1 (E-I1),40 the region -82/-75 5'-TACGTGGT-3' also matched the HRE-consensus sequence.
Transfection assays with ALDA-CAT gene constructs containing IVS-IV
(intron D) sequences and HIF-1 E-I1 promoter LUC gene
constructs in Hep3B cells in concordance with EMSAs showed that neither
the ALDA IVS-IV-HRE nor the HRE of the HIF-1 E-I1
promoter was functionally active.37,40
Indeed, a more detailed inspection of the PAI-1 promoter HRE-1 and
sequences immediately flanking its 5'-region showed that it
contained 3 CA-repeats, which were present also in the 17-mer HRE-1
oligonucleotide used for EMSA (Fig 4A). These CA-repeats 4-6 nt
upstream or downstream from the HRE have been shown to be of importance
in conjunction with the HIF-1 binding sites of the EPO and VEGF genes.
Mutation of the CACA-sequences 3' of the EPO-HRE and also
5' and 3' of VEGF-HRE resulted in a loss of
hypoxia-dependent expression of CAT reporter genes.31,41,35
In addition, such CA repeats in the form of a CACAT-sequence have also
been found 3' from the HRE -204/-181 sequence in the human ALDA
gene promoter37 and in the form of a CACAG sequence 161 bp
in front of the HRE -164/-151 of the rat 1B-adrenergic
receptor gene. Thus, in the PAI-1 promoter, HRE-2 served as the major
HIF-1 binding site, and HRE-1 in conjunction with the CA-repeat was
necessary for the full response of the PAI-1 gene promoter under hypoxia.
Physiological role of the HIF-1-mediated PAI-1 induction.
Hypoxia is an important regulator for vascular homeostasis and a
major stimulus for vascular development. Local hypoxia shifts the
balance between fibrinogenesis and fibrinolysis towards thrombus formation and stabilization42,43 and elicits
neovascularization.44 A key event in these processes is the
local increase in HIF-1. HIF-1, in turn, stimulates the formation of
the fibrinolysis inhibitor or more general extracellular proteolysis
inhibitor PAI-1 as shown in this study and of the angiogenic growth
factor VEGF.45 Inhibition of fibrinolysis by PAI-1 is
important for thrombus formation and stabilization, and stimulation of
growth by VEGF in conjunction with inhibition of extracellular
proteolysis by PAI-1 are important for vessel
formation.43,46,47
PAI-1 in atherosclerosis and thrombosis.
The importance of PAI-1 for atherosclerosis and thrombosis is indicated
by the observation that increased plasma levels of PAI-1 paralleled
reduced fibrinolytic activity of aortic tissue48 in rats
with O2-deprivation (5% O2) and caused fibrin
deposition in the lung of mice exposed to a hypoxic environment (5% to
6% O2),49 and that PAI-1 expression was
enhanced in human atherosclerotic lesions.50-52 Thus, the
HIF-1-mediated activation by hypoxia of the PAI-1 gene, shown here,
may help to explain progression of ischemic coronary heart disease and
fibrin deposition on venous valves during thromboembotic events.
PAI-1 in tumor progression and vascular development.
Hypoxia is a major stimulus for vascular development. The local
increase in HIF-1 in hypoxic areas elicits the formation of the
angiogenic growth factor VEGF45 and as shown in this study of the extracellular proteolysis inhibitor PAI-1. Stimulation of growth
by VEGF and control of proteolysis are important for angiogenesis,53 because low VEGF levels resulted in reduced blood vessel density46 and low levels of PAI-1 in excessive proteolysis preventing coordinated endothelial
sprouting.54,55
The formation of tumors or metastases requires the regulation of
adhesive, proteolytic, as well as migrating events, and finally neovascularization of the tumor or metastasis. Tumors derived from
embryonic stem (ES) cells with inactivated HIF-1 genes
(HIF-1/) did not express VEGF in response to hypoxia
resulting in poor blood vessel supply.56,57 In athymic
mice, tumors derived from injected HepaC4 cells with a defective ARNT
(HIF-1) and thus with a lack of the HIF-1 (HIF-1/HIF-1)
heterodimer had reduced vascularity and grew more slowly than tumors
derived from the wild-type Hepa-1 cells.58,59 Moreover,
after implantation of malignant murine keratinocytes onto the dorsal
muscle fascia of wild-type and PAI-1/ mice, only the
tissues of wild-type mice were infiltrated by the tumor cells. The
failure of tumor cell invasion in PAI-1/ mice was due to
absent vascularization of the tumor, which was restored after rescue of
systemic PAI-1 expression with an adenoviral vector containing the
human PAI-1 gene.47 In line with these observations, PAI-1
was found to be expressed at higher levels in tumor than in other
cells; it may be necessary for optimal metastasis formation as shown
for cultured lung cancer cells.60 Finally, in a number of
clinical studies, it was shown that high PAI-1 levels in patients
suffering from various types of cancer indicated a poor
prognosis.61-63
Thus, it appears that HIF-1 controls production of angiogenic growth
factors, such as VEGF, and as shown in this study, inhibitors of
proteolysis, such as PAI-1, which both contribute to neovascularization and tumor growth.
| |
ACKNOWLEDGMENT |
The authors thank Dr T.D. Gelehrter (Department of Human Genetics,
University Michigan Medical School, Ann Arbor, MI) for the kind gift of
PAI-1 cDNA.
| |
FOOTNOTES |
Submitted December 18, 1998; accepted August 16, 1999.
Supported by the Deutsche Forschungsgemeinschaft SFB 402 Teilprojekt A1.
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 Thomas Kietzmann, MD, Institut
für Biochemie und Molekulare Zellbiologie, Humboldtallee 23, D-37073 Göttingen, Germany; e-mail: tkietzm{at}gwdg.de.
| |
REFERENCES |
1.
Kruithof EK, Tran TC, Bachmann F:
The fast-acting inhibitor of tissue-type plasminogen activator in plasma is also the primary plasma inhibitor of urokinase.
Thromb Haemost
55:65, 1986
[Medline]
[Order article via Infotrieve]
2.
Kruithof EK, Vassalli JD, Schleuning WD, Mattaliano RJ, Bachmann F:
Purification and characterization of a plasminogen activator inhibitor from the histiocytic lymphoma cell line U-937.
J Biol Chem
261:11207, 1986
[Abstract/Free Full Text]
3.
Loskutoff DJ, Sawdey M, Mimuro J:
Type 1 plasminogen activator inhibitor.
Prog Hemost Thromb
9:87, 1989
[Medline]
[Order article via Infotrieve]
4.
Kruithof EK, Baker MS, Bunn CL:
Biological and clinical aspects of plasminogen activator inhibitor type 2.
Blood
86:4007, 1995
[Free Full Text]
5.
Bachmann F:
The enigma PAI-2. Gene expression, evolutionary and functional aspects.
Thromb Haemost
74:172, 1995
[Medline]
[Order article via Infotrieve]
6.
Fearns C, Loskutoff DJ:
Induction of plasminogen activator inhibitor 1 gene expression in murine liver by lipopolysaccharide. Cellular localization and role of endogenous tumor necrosis factor-alpha.
Am J Pathol
150:579, 1997
[Abstract]
7.
Erickson LA, Hekman CM, Loskutoff DJ:
The primary plasminogen-activator inhibitors in endothelial cells, platelets, serum, and plasma are immunologically related.
Proc Natl Acad Sci USA
82:8710, 1985
[Abstract/Free Full Text]
8.
Reilly CF, McFall RC:
Platelet-derived growth factor and transforming growth factor-beta regulate plasminogen activator inhibitor-1 synthesis in vascular smooth muscle cells.
J Biol Chem
266:9419, 1991
[Abstract/Free Full Text]
9.
Le-Magueresse BB, Pernod G, Kolodie L, Morera AM, Benahmed M:
Tumor necrosis factor-alpha regulates plasminogen activator inhibitor-1 in rat testicular peritubular cells.
Endocrinology
138:1097, 1997
[Abstract/Free Full Text]
10.
Busso N, Nicodeme E, Chesne C, Guillouzo A, Belin D, Hyafil F:
Urokinase and type I plasminogen activator inhibitor production by normal human hepatocytes: Modulation by inflammatory agents.
Hepatology
20:186, 1994
[Medline]
[Order article via Infotrieve]
11.
Heaton JH, Nebes VL, O'Dell LG, Morris-SM J, Gelehrter TD:
GLucocorticoid and cyclic nucleotide regulation of plasminogen activator and plasminogen activator-inhibitor gene expression in primary cultures of rat hepatocytes.
Mol Endocrinol
3:185, 1989
[Abstract/Free Full Text]
12.
Podor TJ, Loskutoff DJ:
Immunoelectron microscopic localization of type 1 plasminogen activator inhibitor in the extracellular matrix of transforming growth factor-beta-activated endothelial cells.
Ann N Y Acad Sci
667:46, 1992
[Medline]
[Order article via Infotrieve]
13.
Collen D, Lijnen HR:
Basic and clinical aspects of fibrinolysis and thrombolysis.
Blood
78:3114, 1991
[Free Full Text]
14.
Agrenius V, Chmielewska J, Widstrom O, Blomback M:
Pleural fibrinolytic activity is decreased in inflammation as demonstrated in quinacrine pleurodesis treatment of malignant pleural effusion.
Am Rev Respir Dis
140:1381, 1989
[Medline]
[Order article via Infotrieve]
15.
Eddy AA, Giachelli CM:
Renal expression of genes that promote interstitial inflammation and fibrosis in rats with protein-overload proteinuria.
Kidney Int
47:1546, 1995
[Medline]
[Order article via Infotrieve]
16.
Dieval J, Nguyen G, Gross S, Delobel J, Kruithof EK:
A lifelong bleeding disorder associated with a deficiency of plasminogen activator inhibitor type 1.
Blood
77:528, 1991
[Abstract/Free Full Text]
17.
Fay WP, Shapiro AD, Shih JL, Schleef RR, Ginsburg D:
Brief report: Complete deficiency of plasminogen-activator inhibitor type 1 due to a frame-shift mutation.
N Engl J Med
327:1729, 1992
[Medline]
[Order article via Infotrieve]
18.
Lee MH, Vosburgh E, Anderson K, McDonagh J:
Deficiency of plasma plasminogen activator inhibitor 1 results in hyperfibrinolytic bleeding.
Blood
81:2357, 1993
[Abstract/Free Full Text]
19.
Quax PH, van-den HC, Verheijen JH, Padro T, Zeheb R, Gelehrter TD, van BT, Kuiper J, Emeis JJ:
Endotoxin induction of plasminogen activator and plasminogen activator inhibitor type 1 mRNA in rat tissues in vivo.
J Biol Chem
265:15560, 1990
[Abstract/Free Full Text]
20.
Hamsten A, de FU, Walldius G, Dahlen G, Szamosi A, Landou C, Blomback M, Wiman B:
Plasminogen activator inhibitor in plasma: Risk factor for recurrent myocardial infarction.
Lancet
2:3, 1987
[Medline]
[Order article via Infotrieve]
21.
Nilsson IM, Ljungner H, Tengborn L:
Two different mechanisms in patients with venous thrombosis and defective fibrinolysis: Low concentration of plasminogen activator or increased concentration of plasminogen activator inhibitor.
Br Med J Clin Res Ed
290:1453, 1985
22.
Vaughan DE, Declerck PJ, Van HE, De MM, Collen D:
Reactivated recombinant plasminogen activator inhibitor-1 (rPAI-1) effectively prevents thrombolysis in vivo.
Thromb Haemost
68:60, 1992
[Medline]
[Order article via Infotrieve]
23.
CoLucci M, Paramo JA, Collen D:
Generation in plasma of a fast-acting inhibitor of plasminogen activator in response to endotoxin stimulation.
J Clin Invest
75:818, 1985
24.
Yamashita M, Darlington DN, Weeks EJ, Jones RO, Gann DS:
Plasminogen activator inhibitor-1 rises after hemorrhage in rats.
Am J Physiol
268:E1065, 1995
[Abstract/Free Full Text]
25.
Nauck M, Wolfle D, Katz N, Jungermann K:
Modulation of the glucagon-dependent induction of phosphoenolpyruvate carboxykinase and tyrosine aminotransferase by arterial and venous oxygen concentrations in hepatocyte cultures.
Eur J Biochem
119:657, 1981
[Medline]
[Order article via Infotrieve]
26.
Kietzmann T, Roth U, Freimann S, Jungermann K:
Arterial oxygen partial pressures reduce the insulin-dependent induction of the perivenously located gLucokinase in rat hepatocyte cultures: Mimicry of arterial oxygen pressures by H2O2.
Biochem J
321:17, 1997
27.
Bruzdzinski CJ, Riordan JM, 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]
28.
Immenschuh S, Hinke V, Ohlmann A, Gifhorn-Katz S, Katz N, Jungermann K, Kietzmann T:
Transcriptional activation of the heme oxygenase-1 gene by cGMP via a cAMP response element/activator protein-1 element in primary cultures of rat hepatocytes.
Biochem J
334:141, 1998
29.
Zeheb R, Rafferty UM, Rodriguez MA, Andreasen P, Gelehrter TD:
Immunoaffinity purification of HTC rat hepatoma cell plasminogen activator-inhibitor-1.
Thromb Haemost
58:1017, 1987
[Medline]
[Order article via Infotrieve]
30.
Dignam JD, Lebovitz RM, Roeder RG:
Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei.
Nucleic Acids Res
11:1475, 1983
[Abstract/Free Full Text]
31.
Semenza GL, Wang GL:
A nuclear factor induced by hypoxia via de novo protein synthesis binds to the human erythropoietin gene enhancer at a site required for transcriptional activation.
Mol Cell Biol
12:5447, 1992
[Abstract/Free Full Text]
32.
Kvietikova I, Wenger RH, Marti HH, Gassmann M:
The hypoxia-inducible factor-1 DNA recognition site is cAMP-responsive.
Kidney Int
51:564, 1997
[Medline]
[Order article via Infotrieve]
33.
Bunn HF, Poyton RO:
Oxygen sensing and molecular adaptation to hypoxia.
Physiol Rev
76:839, 1996
[Abstract/Free Full Text]
34.
Semenza GL, Agani F, Booth G, Forsythe J, Iyer N, Jiang BH, Leung S, Roe R, Wiener C, Yu A:
Structural and functional analysis of hypoxia-inducible factor 1.
Kidney Int
51:553, 1997
[Medline]
[Order article via Infotrieve]
35.
Liu Y, Cox SR, Morita T, Kourembanas S:
Hypoxia regulates vascular endothelial growth factor gene expression in endothelial cells. Identification of a 5' enhancer.
Circ Res
77:638, 1995
[Abstract/Free Full Text]
36.
Kvietikova I, Wenger RH, Marti HH, Gassmann M:
The transcription factors ATF-1 and CREB-1 bind constitutively to the hypoxia-inducible factor-1 (HIF-1) DNA recognition site.
Nucleic Acids Res
23:4542, 1995
[Abstract/Free Full Text]
37.
Semenza GL, Jiang BH, Leung SW, Passantino R, Concordet JP, Maire P, Giallongo A:
Hypoxia response elements in the aldolase A, enolase 1, and lactate dehydrogenase A gene promoters contain essential binding sites for hypoxia-inducible factor 1.
J Biol Chem
271:32529, 1996
[Abstract/Free Full Text]
38.
Firth JD, Ebert BL, Pugh CW, Ratcliffe PJ:
Oxygen-regulated control elements in the phosphoglycerate kinase 1 and lactate dehydrogenase A genes: Similarities with the erythropoietin 3' enhancer.
Proc Natl Acad Sci USA
91:6496, 1994
[Abstract/Free Full Text]
39.
Rolfs A, Kvietikova I, Gassmann M, Wenger RH:
Oxygen-regulated transferrin expression is mediated by hypoxia-inducible factor-1.
J Biol Chem
272:20055, 1997
[Abstract/Free Full Text]
40.
Wenger RH, Rolfs A, Kvietikova I, Spielmann P, Zimmermann DR, Gassmann M:
The mouse gene for hypoxia-inducible factor-1alpha genomic organization, expression and characterization of an alternative first exon and 5' flanking sequence.
Eur J Biochem
246:155, 1997
[Medline]
[Order article via Infotrieve]
41.
Beck I, Weinmann R, Caro J:
Characterization of hypoxia-responsive enhancer in the human erythropoietin gene shows presence of hypoxia-inducible 120-Kd nuclear DNA-binding protein in erythropoietin-producing and nonproducing cells.
Blood
82:704, 1993
[Abstract/Free Full Text]
42.
Carmeliet P, Collen D:
Gene targeting and gene transfer studies of the plasminogen/plasmin system: Implications in thrombosis, hemostasis, neointima formation, and atherosclerosis.
FASEB J
9:934, 1995
[Abstract]
43.
Carmeliet P, Collen D:
Vascular development and disorders: Molecular analysis and pathogenic insights.
Kidney Int
53:1519, 1998
[Medline]
[Order article via Infotrieve]
44.
Semenza GL:
Hypoxia-inducible factor 1 and the molecular physiology of oxygen homeostasis.
J Lab Clin Med
131:207, 1998
[Medline]
[Order article via Infotrieve]
45.
Forsythe JA, Jiang BH, Iyer NV, Agani F, Leung SW, Koos RD, Semenza GL:
Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1.
Mol Cell Biol
16:4604, 1996
[Abstract]
46.
Carmeliet P, Ferreira V, Breier G, Pollefeyt S, Kieckens L, Gertsenstein M, Fahrig M, Vandenhoeck A, Harpal K, Eberhardt C, Declercq C, Pawling J, Moons L, Collen D, Risau W, Nagy A:
Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele.
Nature
380:435, 1996
[Medline]
[Order article via Infotrieve]
47.
Bajou K, Noel A, Gerard RD, Masson V, Brunner N, Holst-Hansen C, Skobe M, Fusenig NE, Carmeliet P, Collen D, Foidart JM:
Absence of host plasminogen activator inhibitor 1 prevents cancer invasion and vascularization.
Nat Med
4:923, 1998
[Medline]
[Order article via Infotrieve]
48.
Risberg B, Stenberg B:
Modulation of tissue fibrinolysis from hypoxia and hyperoxia.
Thromb Res
38:129, 1985
[Medline]
[Order article via Infotrieve]
49.
Pinsky DJ, Liao H, Lawson CA, Yan SF, Chen JX, Carmeliet P, Loskutoff DL, Stern DM:
Coordinated induction of plasminogen activator inhibitor-1 (PAI-1) and inhibition of plasminogen activator gene expression by hypoxia promotes pulmonary vascular fibrin deposition.
J Clin Invest
102:919, 1998
[Medline]
[Order article via Infotrieve]
50.
Schneiderman J, Sawdey MS, Keeton MR, Bordin GM, Bernstein EF, Dilley RB, Loskutoff DJ:
Increased type 1 plasminogen activator inhibitor gene expression in atherosclerotic human arteries.
Proc Natl Acad Sci USA
89:6998, 1992
[Abstract/Free Full Text]
51.
Lupu F, Bergonzelli GE, Heim DA, Cousin E, Genton CY, Bachmann F, Kruithof EK:
Localization and production of plasminogen activator inhibitor-1 in human healthy and atherosclerotic arteries.
Arterioscler Thromb
13:1090, 1993
[Abstract/Free Full Text]
52.
Robbie LA, Booth NA, Brown AJ, Bennett B:
Inhibitors of fibrinolysis are elevated in atherosclerotic plaque.
Arterioscler Thromb Vasc Biol
16:539, 1996
[Abstract/Free Full Text]
53.
Schnaper HW, Barnathan ES, Mazar A, Maheshwari S, Ellis S, Cortez SL, Baricos WH, Kleinman HK:
Plasminogen activators augment endothelial cell organization in vitro by two distinct pathways.
J Cell Physiol
165:107, 1995
[Medline]
[Order article via Infotrieve]
54.
Pepper MS, Sappino AP, Montesano R, Orci L, Vassalli JD:
Plasminogen activator inhibitor-1 is induced in migrating endothelial cells.
J Cell Physiol
153:129, 1992
[Medline]
[Order article via Infotrieve]
55.
Montesano R, Pepper MS, Mohle SU, Risau W, Wagner EF, Orci L:
Increased proteolytic activity is responsible for the aberrant morphogenetic behavior of endothelial cells expressing the middle T oncogene.
Cell
62:435, 1990
[Medline]
[Order article via Infotrieve]
56.
Ryan HE, Lo J, Johnson RS:
HIF-1 alpha is required for solid tumor formation and embryonic vascularization.
EMBO J
17:3005, 1998
[Medline]
[Order article via Infotrieve]
57.
Carmeliet P, Dor Y, Herbert JM, Fukumura D, Brusselmans K, Ewerchin D, Neeman M, Bono F, Abramovitch R, Maxwell P, Koch CJ, Ratcliffe P, Moons L, Jain RK, Collen D, Keshet E:
Role of HIF-1 alpha or in hypoxia-mediated apoptosis, cell proliferation and tumour angiogenesis.
Nature
394:485, 1998
[Medline]
[Order article via Infotrieve]
58.
Maxwell PH, Dachs GU, Gleadle JM, Nicholls LG, Harris AL, Stratford IJ, Hankinson O, Pugh CW, Ratcliffe PJ:
Hypoxia-inducible factor-1 modulates gene expression in solid tumors and influences both angiogenesis and tumor growth.
Proc Natl Acad Sci USA
94:8104, 1997
[Abstract/Free Full Text]
59.
Jiang BH, Agani F, Passaniti A, Semenza GL:
V-SRC induces expression of hypoxia-inducible factor 1 (HIF-1) and transcription of genes encoding vascular endothelial growth factor and enolase 1: Involvement of HIF-1 in tumor progression.
Cancer Res
57:5328, 1997
[Abstract/Free Full Text]
60.
Liu G, Shuman MA, Cohen RL:
Co-expression of urokinase, urokinase receptor and PAI-1 is necessary for optimum invasiveness of cultured lung cancer cells.
Int J Cancer
60:501, 1995
[Medline]
[Order article via Infotrieve]
61.
Pedersen H, Brunner N, Francis D, Osterlind K, Ronne E, Hansen HH, Dano K, Grondahl HJ:
Prognostic impact of urokinase, urokinase receptor, and type 1 plasminogen activator inhibitor in squamous and large cell lung cancer tissue.
Cancer Res
54:4671, 1994
[Abstract/Free Full Text]
62.
Grondahl HJ, Christensen IJ, Rosenquist C, Brunner N, Mouridsen HT, Dano K, Blichert TM:
High levels of urokinase-type plasminogen activator and its inhibitor PAI-1 in cytosolic extracts of breast carcinomas are associated with poor prognosis.
Cancer Res
53:2513, 1993
[Abstract/Free Full Text]
63.
Kuhn W, Pache L, Schmalfeldt B, Dettmar P, Schmitt M, Janicke F, Graeff H:
Urokinase (uPA) and PAI-1 predict survival in advanced ovarian cancer patients (FIGO III) after radical surgery and platinum-based chemotherapy.
Gynecol Oncol
55:401, 1994
[Medline]
[Order article via Infotrieve]
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
M. D. Michaud, G. A. Robitaille, J.-P. Gratton, and D. E. Richard
Sphingosine-1-Phosphate: A Novel Nonhypoxic Activator of Hypoxia-Inducible Factor-1 in Vascular Cells
Arterioscler. Thromb. Vasc. Biol.,
June 1, 2009;
29(6):
902 - 908.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J.-O. Moon, T. P. Welch, F. J. Gonzalez, and B. L. Copple
Reduced liver fibrosis in hypoxia-inducible factor-1{alpha}-deficient mice
Am J Physiol Gastrointest Liver Physiol,
March 1, 2009;
296(3):
G582 - G592.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Kimura, M. Iwano, D. F. Higgins, Y. Yamaguchi, K. Nakatani, K. Harada, A. Kubo, Y. Akai, E. B. Rankin, E. G. Neilson, et al.
Stable expression of HIF-1{alpha} in tubular epithelial cells promotes interstitial fibrosis
Am J Physiol Renal Physiol,
October 1, 2008;
295(4):
F1023 - F1029.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Klein, D. Flugel, and T. Kietzmann
Transcriptional Regulation of Serine/Threonine Kinase-15 (STK15) Expression by Hypoxia and HIF-1
Mol. Biol. Cell,
September 1, 2008;
19(9):
3667 - 3675.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Gross, G. Buchwalter, H. Dubois-Pot, E. Cler, H. Zheng, and B. Wasylyk
The Ternary Complex Factor Net Is Downregulated by Hypoxia and Regulates Hypoxia-Responsive Genes
Mol. Cell. Biol.,
June 1, 2007;
27(11):
4133 - 4141.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Wouters, B. Pauwels, F. Lardon, and J. B. Vermorken
Review: Implications of In Vitro Research on the Effect of Radiotherapy and Chemotherapy Under Hypoxic Conditions
Oncologist,
June 1, 2007;
12(6):
690 - 712.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. Flugel, A. Gorlach, C. Michiels, and T. Kietzmann
Glycogen Synthase Kinase 3 Phosphorylates Hypoxia-Inducible Factor 1{alpha} and Mediates Its Destabilization in a VHL-Independent Manner
Mol. Cell. Biol.,
May 1, 2007;
27(9):
3253 - 3265.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Bonello, C. Zahringer, R. S. BelAiba, T. Djordjevic, J. Hess, C. Michiels, T. Kietzmann, and A. Gorlach
Reactive Oxygen Species Activate the HIF-1{alpha} Promoter Via a Functional NF{kappa}B Site
Arterioscler. Thromb. Vasc. Biol.,
April 1, 2007;
27(4):
755 - 761.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Q. Ke and M. Costa
Hypoxia-Inducible Factor-1 (HIF-1)
Mol. Pharmacol.,
November 1, 2006;
70(5):
1469 - 1480.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. A. Russell
Management of Sepsis
N. Engl. J. Med.,
October 19, 2006;
355(16):
1699 - 1713.
[Full Text]
[PDF]
|
|
|
|
|
|
|
V. H. Haase
Hypoxia-inducible factors in the kidney
Am J Physiol Renal Physiol,
August 1, 2006;
291(2):
F271 - F281.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
V. A. Carroll and M. Ashcroft
Role of Hypoxia-Inducible Factor (HIF)-1{alpha} versus HIF-2{alpha} in the Regulation of HIF Target Genes in Response to Hypoxia, Insulin-Like Growth Factor-I, or Loss of von Hippel-Lindau Function: Implications for Targeting the HIF Pathway.
Cancer Res.,
June 15, 2006;
66(12):
6264 - 6270.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. P. Luyendyk, L. D. Lehman-McKeeman, D. M. Nelson, V. M. Bhaskaran, T. P. Reilly, B. D. Car, G. H. Cantor, X. Deng, J. F. Maddox, P. E. Ganey, et al.
Coagulation-Dependent Gene Expression and Liver Injury in Rats Given Lipopolysaccharide with Ranitidine but Not with Famotidine
J. Pharmacol. Exp. Ther.,
May 1, 2006;
317(2):
635 - 643.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. N. Cheema, T. Hong, N. Nili, A. Segev, J. G. Moffat, K. E. Lipson, A. R. Howlett, D. W. Holdsworth, M. J. Cole, B. Qiang, et al.
Adventitial Microvessel Formation After Coronary Stenting and the Effects of SU11218, a Tyrosine Kinase Inhibitor
J. Am. Coll. Cardiol.,
March 7, 2006;
47(5):
1067 - 1075.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
U. Danilczyk and J. M. Penninger
Angiotensin-Converting Enzyme II in the Heart and the Kidney
Circ. Res.,
March 3, 2006;
98(4):
463 - 471.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. Y. Dimova and T. Kietzmann
Cell Type-dependent Regulation of the Hypoxia-responsive Plasminogen Activator Inhibitor-1 Gene by Upstream Stimulatory Factor-2
J. Biol. Chem.,
February 3, 2006;
281(5):
2999 - 3005.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. Nakamura, P. Abreu-e-Lima, Y. Awakura, T. Inoue, T. Kamoto, O. Ogawa, H. Kotani, T. Manabe, G.-J. Zhang, K. Kondo, et al.
Clusterin Is a Secreted Marker for a Hypoxia-Inducible Factor-Independent Function of the von Hippel-Lindau Tumor Suppressor Protein
Am. J. Pathol.,
February 1, 2006;
168(2):
574 - 584.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. E. Lash, H. A. Otun, B. A. Innes, J. N. Bulmer, R. F. Searle, and S. C. Robson
Low Oxygen Concentrations Inhibit Trophoblast Cell Invasion from Early Gestation Placental Explants via Alterations in Levels of the Urokinase Plasminogen Activator System
Biol Reprod,
February 1, 2006;
74(2):
403 - 409.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. Buchwalter, C. Gross, and B. Wasylyk
The Ternary Complex Factor Net Regulates Cell Migration through Inhibition of PAI-1 Expression
Mol. Cell. Biol.,
December 15, 2005;
25(24):
10853 - 10862.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Hayashi, M. Sakata, T. Takeda, M. Tahara, T. Yamamoto, Y. Okamoto, R. Minekawa, A. Isobe, M. Ohmichi, K. Tasaka, et al.
Up-Regulation of c-met Protooncogene Product Expression through Hypoxia-Inducible Factor-1{alpha} Is Involved in Trophoblast Invasion under Low-Oxygen Tension
Endocrinology,
November 1, 2005;
146(11):
4682 - 4689.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. H. Wenger, D. P. Stiehl, and G. Camenisch
Integration of Oxygen Signaling at the Consensus HRE
Sci. Signal.,
October 18, 2005;
2005(306):
re12 - re12.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Zhou, S. Lechpammer, J. S. Greenberger, and J. Glowacki
Hypoxia Inhibition of Adipocytogenesis in Human Bone Marrow Stromal Cells Requires Transforming Growth Factor-{beta}/Smad3 Signaling
J. Biol. Chem.,
June 17, 2005;
280(24):
22688 - 22696.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Q. Liu, U. Moller, D. Flugel, and T. Kietzmann
Induction of plasminogen activator inhibitor I gene expression by intracellular calcium via hypoxia-inducible factor-1
Blood,
December 15, 2004;
104(13):
3993 - 4001.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. F. Higgins, M. P. Biju, Y. Akai, A. Wutz, R. S. Johnson, and V. H. Haase
Hypoxic induction of Ctgf is directly mediated by Hif-1
Am J Physiol Renal Physiol,
December 1, 2004;
287(6):
F1223 - F1232.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Sato, T. Tanaka, K. Maemura, T. Uchiyama, H. Sato, T. Maeno, T. Suga, T. Iso, Y. Ohyama, M. Arai, et al.
The PAI-1 Gene as a Direct Target of Endothelial PAS Domain Protein-1 in Adenocarcinoma A549 Cells
Am. J. Respir. Cell Mol. Biol.,
August 1, 2004;
31(2):
209 - 215.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. P. Luyendyk, W. B. Mattes, L. D. Burgoon, T. R. Zacharewski, J. F. Maddox, G. N. Cosma, P. E. Ganey, and R. A. Roth
Gene Expression Analysis Points to Hemostasis in Livers of Rats Cotreated with Lipopolysaccharide and Ranitidine
Toxicol. Sci.,
July 1, 2004;
80(1):
203 - 213.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y. Wu, Q. Zhang, D. K. Ann, A. Akhondzadeh, H. S. Duong, D. V. Messadi, and A. D. Le
Increased vascular endothelial growth factor may account for elevated level of plasminogen activator inhibitor-1 via activating ERK1/2 in keloid fibroblasts
Am J Physiol Cell Physiol,
April 1, 2004;
286(4):
C905 - C912.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. Pichiule, J. C. Chavez, and J. C. LaManna
Hypoxic Regulation of Angiopoietin-2 Expression in Endothelial Cells
J. Biol. Chem.,
March 26, 2004;
279(13):
12171 - 12180.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Q. Liu, U. Berchner-Pfannschmidt, U. Moller, M. Brecht, C. Wotzlaw, H. Acker, K. Jungermann, and T. Kietzmann
A Fenton reaction at the endoplasmic reticulum is involved in the redox control of hypoxia-inducible gene expression
PNAS,
March 23, 2004;
101(12):
4302 - 4307.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. C. Blouin, E. L. Page, G. M. Soucy, and D. E. Richard
Hypoxic gene activation by lipopolysaccharide in macrophages: implication of hypoxia-inducible factor 1{alpha}
Blood,
February 1, 2004;
103(3):
1124 - 1130.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
U. Roth, K. Curth, T. G. Unterman, and T. Kietzmann
The Transcription Factors HIF-1 and HNF-4 and the Coactivator p300 Are Involved in Insulin-regulated Glucokinase Gene Expression via the Phosphatidylinositol 3-Kinase/Protein Kinase B Pathway
J. Biol. Chem.,
January 23, 2004;
279(4):
2623 - 2631.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. O. Bates and R. O. P. Jones
The Role of Vascular Endothelial Growth Factor in Wound Healing
International Journal of Lower Extremity Wounds,
June 1, 2003;
2(2):
107 - 120.
[Abstract]
[PDF]
|
|
|
|
|
|
|
T. Kietzmann, A. Samoylenko, and S. Immenschuh
Transcriptional Regulation of Heme Oxygenase-1 Gene Expression by MAP Kinases of the JNK and p38 Pathways in Primary Cultures of Rat Hepatocytes
J. Biol. Chem.,
May 9, 2003;
278(20):
17927 - 17936.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Kietzmann, A. Samoylenko, U. Roth, and K. Jungermann
Hypoxia-inducible factor-1 and hypoxia response elements mediate the induction of plasminogen activator inhibitor-1 gene expression by insulin in primary rat hepatocytes
Blood,
February 1, 2003;
101(3):
907 - 914.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
W. Dichtl, J. Dulak, M. Frick, H. F. Alber, S. P. Schwarzacher, M. P.S. Ares, J. Nilsson, O. Pachinger, and F. Weidinger
HMG-CoA Reductase Inhibitors Regulate Inflammatory Transcription Factors in Human Endothelial and Vascular Smooth Muscle Cells
Arterioscler. Thromb. Vasc. Biol.,
January 1, 2003;
23(1):
58 - 63.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. H. WENGER
Cellular adaptation to hypoxia: O2-sensing protein hydroxylases, hypoxia-inducible transcription factors, and O2-regulated gene expression
FASEB J,
August 1, 2002;
16(10):
1151 - 1162.
[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]
|
|
|
|
|
|
|
T. Fink, A. Kazlauskas, L. Poellinger, P. Ebbesen, and V. Zachar
Identification of a tightly regulated hypoxia-response element in the promoter of human plasminogen activator inhibitor-1
Blood,
March 15, 2002;
99(6):
2077 - 2083.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. M. Providence, L. A. White, J. Tang, J. Gonclaves, L. Staiano-Coico, and P. J. Higgins
Epithelial monolayer wounding stimulates binding of USF-1 to an E-box motif in the plasminogen activator inhibitor type 1 gene
J. Cell Sci.,
January 10, 2002;
115(19):
3767 - 3777.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Krones, K. Jungermann, and T. Kietzmann
Cross-Talk between the Signals Hypoxia and Glucose at the Glucose Response Element of the L-Type Pyruvate Kinase Gene
Endocrinology,
June 1, 2001;
142(6):
2707 - 2718.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Samoylenko, U. Roth, K. Jungermann, and T. Kietzmann
The upstream stimulatory factor-2a inhibits plasminogen activator inhibitor-1 gene expression by binding to a promoter element adjacent to the hypoxia-inducible factor-1 binding site
Blood,
May 1, 2001;
97(9):
2657 - 2666.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. Wenger
Mammalian oxygen sensing, signalling and gene regulation
J. Exp. Biol.,
January 4, 2000;
203(8):
1253 - 1263.
[Abstract]
[PDF]
|
|
|
|
|
|
|
A. Gorlach, I. Diebold, V. B. Schini-Kerth, U. Berchner-Pfannschmidt, U. Roth, R. P. Brandes, T. Kietzmann, and R. Busse
Thrombin Activates the Hypoxia-Inducible Factor-1 Signaling Pathway in Vascular Smooth Muscle Cells : Role of the p22phox-Containing NADPH Oxidase
Circ. Res.,
July 6, 2001;
89(1):
47 - 54.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
TOP
ABSTRACT
INTRODUCTION