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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Danish Cancer Society, Department of Virus and
Cancer, Aarhus, Denmark; the Department of Cell and Molecular Biology,
Medical Nobel Institute, Karolinska Institutet, Stockholm, Sweden; and
the University of Aalborg, Denmark.
Plasminogen activator inhibitor-1 (PAI-1) plays a key role in
control of coagulation and tissue remodeling and has been shown to be
regulated by a number of cell stimuli, among those hypoxia. In this
study we characterize the hypoxia-mediated induction of PAI-1 in human
hepatoma cell line HepG2. We found that PAI-1 is tightly regulated in a
narrow oxygen gradient. After incubation at oxygen concentrations of
1% to 2%, a 60-fold increase in PAI-1 messenger RNA levels was
observed, whereas mild hypoxic conditions of more than 3.5% did not
appear to induce transcription. Moreover, increased levels of PAI-1
protein were observed after incubation at low oxygen tensions. Through
sequence analysis, several putative hypoxia-response elements (HREs
1-5) were identified in the human PAI-I promoter. Reporter gene assays
showed that the HRE-2 ( Plasminogen activator inhibitor-1 (PAI-1) plays a
central role in the control of physiologically important mechanisms
involved in the homeostasis of blood coagulation and remodeling of
extracellular matrix (reviewed by Booth1). The effect of
PAI-1 is mediated through inhibition of urokinase and tissue type
plasminogen activators. The importance of PAI-1 in the regulation of
fibrinolytic activity is highlighted by several studies documenting an
association between increased levels of PAI-1 and the risk of
developing a cardiovascular disease.2-4 Furthermore,
numerous clinical studies of different types of cancer have identified
high levels of plasma PAI-1 as a strong prognostic factor for more
metastatic forms of cancer, concomitant with a poorer clinical outcome
(reviewed by Harbeck et al5).
PAI-1 is produced by a variety of cell types in vitro, such as
hepatocytes,6 platelets,7 smooth muscle
cells,8 and endothelial cells.9 The sources
of PAI-1 in vivo have not as yet been identified. However, studies in
rabbits indicate that the liver and endothelial cells are the most
important producers.10 Several agents induce PAI-1 at the
transcriptional level, including phorbol esters,11
inflammatory cytokines,12 transforming growth factor
We have previously found that PAI-1 was up-regulated in 4 different
human liver cell lines on incubation in 1% oxygen.19 In
the present study, we have characterized hypoxia-induced expression of
PAI-1 in the human hepatoma cell line HepG2 cultured in a controlled atmosphere with oxygen concentrations ranging from 1% to 8%.
Moreover, we have identified an element in the human PAI-1 promoter
mediating hypoxic responses and demonstrated that this element binds
HIF-1.
Cell cultures
Hypoxic treatment
Real time reverse transcriptase-polymerase chain reaction For preparation of RNA, the GenElute messenger RNA (mRNA) kit (Sigma, St Louis, MO) was used according to the manufacturer's instructions. Briefly, the medium was withdrawn from one well, and the cells were lysed by the addition of 300 µL lysis buffer. The lysate was collected and stored at 80°C until completion of the
experiment, when all samples were processed for RNA extraction simultaneously. After isolation, the RNA was DnaseI treated and used
for complementary DNA (cDNA) synthesis. The cDNA was prepared from
approximately 2 µg RNA by using the M-MLV reverse transcriptase (Sigma) with random decamer primers. The levels of PAI-1 transcript were determined by real-time reverse transcriptase-polymerase chain
reaction (RT-PCR) as described.19 To normalize for input load of cDNA between the samples, 18S rRNA was used as an external endogenous standard. The forward, 5'-AGGACCGCGGTTCTATTTTGTTGG-3', and
reverse, 5'-CCCCCGGCCGTCCCTCTTA-3', primers for 18S rRNA were designed
by using the PrimerSelect program of the Lasergene software package
(DNASTAR, Madison, WI) and were used at a concentration of 1 pmol per
reaction. The amplification was performed in an iCycler (Bio-Rad,
Hercules, CA), using a 2-temperature cycling, consisting of a
denaturation step for 15 seconds at 95°C and an annealing/extension
step for 30 seconds at 68°C. For the detection of the PCR products in
real-time, the SYBR Green I fluorophore (Molecular Probes, Leiden, The
Netherlands) was used in a final 22 000-fold dilution from the stock
supplied by the manufacturer. For quantitative analysis of the PAI-1
transcripts and 18S rRNA, the cDNA from each sample was analyzed in
duplicate and on 2 separate occasions.
Quantitation of cell- and matrix-associated and soluble PAI-1 For immunoblotting, the cells were lysed in SDS sample buffer containing 1 mM phenylmethylsulfonyl fluoride and protease inhibitors (Complete; Roche, Mannheim, DE), and the protein concentration was determined by NanoOrange (Molecular Probes). Protein (20 µg) was loaded onto a 10% SDS-polyacrylamide gel and after electrophoresis was blotted onto a polyvinylidene diflouride membrane (Millipore, Bedford, MA). Proteins were stained with Sypro Ruby (Bio-Rad), detected in a Flour-S MultiImager (Bio-Rad), and analyzed by using the TotalLab software package (Phoretix, Newcastle, United Kingdom) to normalize for differences between samples in protein load and transfer. The detection of PAI-1 was carried out by using a 1000-fold dilution of primary rabbit antibody against human PAI-1 (Santa Cruz Biotechnology, Santa Cruz, CA) and a 50 000-fold dilution of biotinylated secondary antibody (goat antirabbit immunoglobulin G; DAKO, Copenhagen, Denmark). Finally, 60 000-fold diluted horseradish peroxidase-conjugated streptavidin (Amersham, Uppsala, Sweden) was used, and, for visualization, the SuperSignal West Femto substrate (Pierce, Rockford, IL) was used. The detection was performed in the Fluor-S MultiImager, and the signals were analyzed by using the TotalLab software package. For each sample the Western blotting procedure was performed at least twice. The concentration of secreted PAI-1 in the medium was determined by enzyme-linked immunosorbent assay (Imulyse PAI-1, Biopool, Ventura, CA), and each sample was analyzed in duplicate.Plasmid constructs, transfection procedure, and reporter assay The human PAI-1 promotor region, extending from positions806
to +19 was amplified from a pEMBL8cat plasmid containing the PAI-promotor (a kind gift from P. Andreasen, University of Aarhus, Denmark) with the aid of an upstream and downstream primer
incorporating restriction sites for MluI and
BglII, respectively. The sequence of the upstream primer was
5'-TGAACGCGTAAGCTTTTACCATGGTAACCCCT-3' and that of the downstream
primer was 5'-TGAAGATCTGCAGCCAAACACAGCTGTGCT-3'. After restriction, the
PCR product was ligated into the luciferase reporter plasmid, pGL3basic
(Promega, Madison, WI), yielding a pGL3-PAI-wt12 345 construct
containing the 5 putative hypoxia-response element (HRE) motifs (Figure
3A). The individual HRE motifs were mutated by using a QuickChange
Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA), and the
mutation primers are listed in Table 1. The generated plasmids were designated pGL3-PAI-wtx, where x denotes the presence of wild-type putative HREs.
HepG2 cells were transfected with the pGL3-PAI-wtx constructs. Briefly, 1.5 × 106 cells in 400 µL growth medium were mixed with 10 µg plasmid DNA and electroporated (220 V, 800 µF) by using the Gene Pulser II system (Bio-Rad). The cells were then seeded in 200 µL medium in 96-well plates in sixplicate at a concentration of 50 × 103 cells/well and incubated for 3 hours at 21% O2, after which half of the plates were transferred to the hypoxic chamber and incubated for 24 hours before analysis. The luciferase activity was determined by using the Steady-Glo luciferase assay (Promega) according to the manufacturer's instructions. Preparation of nuclear extracts For nuclear extract preparation, HepG2 cells in 10-cm dishes were cultured at 1% or 21% oxygen for 6 hours. The cells were then washed twice with ice-cold phosphate-buffered saline (PBS), scraped into 10 mL PBS and pelleted by centrifugation at 2000 rpm for 5 minutes at 4°C. The cell pellet was resuspended in hypotonic buffer HB (10 mM Tris-HCl [pH 7.3], 10 mM KCl, 1.5 mM MgCl2, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol), incubated on ice for 10 minutes and pelleted by centrifugation at 1500 rpm for 5 minutes at 4°C. The cell pellet was resuspended in lysis buffer (HB with 0.4% NP-40) and incubated on ice for 10 minutes. The nuclei were pelleted by centrifugation at 1500 rpm for 5 minutes at 4°C and washed once in HB. The nuclear proteins were extracted by incubation in high-salt buffer (20 mM Tris-HCl [pH 7.3], 25% glycerol, 1.5 mM MgCl2, 0.2 mM EDTA, 0.42 M KCl, 20 µg/mL leupeptin, 1 µg/mL pepstatin) for 20 minutes on ice. The nuclear debris was pelleted by centrifugation at 14 000 rpm for 20 minutes at 4°C. The total protein concentration was determined by the Bradford method.Alignment of human and rat promoter Alignment of the promoters was performed by using the Megalign program (DNASTAR).Electrophoretic mobility shift assay Sequences of probes used for electrophoretic mobility shift assay (EMSA) encompassing the putative HRE motifs and mutant probes are shown in Table 1. The probes were end-labeled with T4 polynucleotide kinase and -32P]-ATP and purified on G25 Microspin
columns (Pharmacia). DNA binding reactions were carried out with 12 µg of proteins, 6-fmol labeled probe in 20 µL reaction volume with
final concentrations of 10 mM Hepes, 2.8 mM MgCl2, 16%
glycerol, 0.15 mM EDTA, 0.25 mM dithiothreitol, 0.1 M KCl, 5 mM
Tris-HCl [pH 7.3], 1 µg poly(dI-dC), and 1 µg poly(dC). The
reactions were incubated on ice for 30 minutes. In inhibition
experiments cold competitor probe was added at 5-, 50-, and 500-fold
molar excess. In control experiments, HIF-1 and ARNT
proteins were expressed by in vitro translation in rabbit reticulocyte
lysate (Promega) by using plasmids pSP72/HIF-121 and
pGEM7/Arnt22 for HIF-1 and ARNT, respectively. For
supershift experiments, polyclonal antibodies against HIF-1 and its
partner factor ARNT were added after an initial 30-minute incubation of the probe with nuclear extract and incubated for 1 hour at 4°C. The
complexes were resolved on a 4% polyacrylamide gel in a
Tris-glycine-EDTA buffer at 30 mA at 4°C.
Hypoxia-induced expression of PAI-1 HepG2 cells were cultured for up to 48 hours under different oxygen concentrations, ranging from 1% to 8%, and at ambient air conditions. As shown in Figure 1A, oxygen concentrations of 1% and 2% rapidly induced PAI-1 mRNA expression leading to statistically significant (P < .05) increases after only 2 hours and reaching maximal levels of a 60-fold induction response after 32 hours of hypoxic treatment. At oxygen concentrations from 3.5% to 5%, a scant 5-fold induction of PAI-1 mRNA expression was observed after 40 hours of hypoxic treatment, whereas no changes in PAI-1 mRNA levels could be observed at oxygen tensions above 6.5%, relative to the levels observed in the ambient air control samples.
To establish whether the increased mRNA levels resulted in any increase in PAI-I protein levels, the intracellular/matrix-associated and soluble levels of PAI-1 protein were determined by immunoblotting (Figures 1B,C) and enzyme-linked immunosorbent assay (Figure 1D), respectively. In the case of cells cultured at 1% and 2% O2, an increase in intracellular and matrix-associated PAI-1 content was evident after 8 hours (Figure 1B), and the levels reached a maximum of an approximately 7-fold increase over the levels detected at ambient air controls after 24 hours of incubation (Figure 1C). In the case of cells cultured at oxygen concentrations ranging from 3.5% to 8%, only moderate increases (approximately 2-fold) over to ambient air controls were observed. In addition, the levels of soluble PAI-1 protein were determined (Figure 1D). The increase in intracellular and matrix-bound PAI-1 protein levels preceded that of soluble PAI-1 by 8 hours. PAI-1 was secreted continuously for the duration of the experiment. After 48 hours of hypoxic treatment at 1% and 2% oxygen, the secreted levels of PAI-1 were approximately 5-fold higher than those for the ambient controls. The cells cultured at 3.5% to 5% secreted twice as much as controls, whereas the secretion of PAI-1 by cells cultured at 6.5% oxygen or more was comparable to that of the control cells. Identification of the hypoxia-responsive element in the human PAI-1 promotor Sequence analysis of the human PAI-1 promotor revealed 5 putative HRE motifs, showing homology with the HIF-1 binding consensus sequence BACGTSSK (B = G/C/T, S = G/C, and K = G/T).23 The first potential HIF-1 binding site, HRE-1, at positions158 to 151, the second site, HRE-2 (positions 194 to 187), and the third site,
HRE-3 (positions 453 to 446), shared homology with the consensus
sequence in 6 of 8 bases, the fourth site, HRE-4 (positions 566 to
559), in 7 of 8 bases, and, finally, the fifth site, HRE-5 (positions
681 to 674), in all 8 bases (Figure
2). Alignment with the rat PAI-1 promoter
showed that the HRE-1 site corresponding to the HIF-1 binding site of
the rat promoter24 deviated in position 4 from the
consensus sequence and that there was complete conservation between the
HRE-2 site and the site identified in the rat promoter as an upstream
stimulatory factor 2a binding site (Figure 2).25 None of
the other motifs shared complete conservation with the rat promoter,
however. In fact, motifs 4 and 5 contained the E box sequence
CACGTG.
Transfection experiments with the wild-type promoter (Figure
3A) and 5 mutant constructs in which the
putative HRE motifs were individually mutated revealed that the HRE-2
was necessary for hypoxia-dependent activation (Figure 3B). Mutations
of the HRE-1 and -3 motifs did not affect the luciferase activity.
Finally, when the HRE-4 or -5 motifs were mutated, the basal levels of luciferase activity were decreased (data not shown). However, this
decrease did not affect hypoxia-dependent activation of promoter activity. Transfection experiments in which the mutant constructs contained only one wild-type putative HRE demonstrated that the HRE-2
motif was sufficient to mediate the maximal response (Figure 3C). The
transfection experiments with the construct in which all putative HRE
motifs were mutated showed no hypoxia-dependent activation response,
demonstrating that no other functional HRE was present in the promoter.
Analysis of DNA binding activity We next performed protein-DNA interaction studies with the sequences spanning the individual HRE motifs. To this end protein-DNA interaction was assessed by EMSA using nuclear extracts obtained from cells incubated at ambient air or under hypoxic conditions (1% O2) for 6 hours. In the case of probes containing the HRE-1, -4, and -5 motifs, a distinctive constitutive, hypoxia-independent complex was formed. This complex was not formed with any of the mutated probes (Figure 4A). When the probe containing HRE-2 was incubated with nuclear extract from hypoxic cells, 2 distinct complexes were formed, a constitutive and a hypoxia-inducible one. The hypoxia-dependent complex was not detected on mutation of the core sequence 5'-CACGTACA-3' to 5'-CTTAATCA-3', demonstrating that the HRE consensus sequence was essential for hypoxia-induced complex formation. Finally, we observed no complex formation with the probes containing either the wild-type or mutated HRE-3 motifs (Figure 4A). The specificity of the binding was verified by adding increasing amounts of cold probe, which competed for binding of the complex, whereas addition of mutated probe did not influence the formation of the hypoxia-inducible complex (Figure 4B). As the HRE-2 differs from the consensus HIF-1 binding sequence with respect to 2 nucleotides, gel shift experiments were performed by using in vitro-translated HIF-1
and ARNT and wild-type HRE-2 probe. As shown in Figure 4C, when either
HIF-1 or ARNT were incubated with the probe, as expected no specific
complexes were formed. However, when HIF-1 and ARNT were allowed to
dimerize, a specific complex was created. This finding indicated that
the HRE-2 sequence was capable of binding the HIF-1-ARNT
heterodimer. Finally, the identity of the hypoxia-dependent DNA-protein
complex generated with nuclear extract was confirmed in a supershift
analysis (Figure 4D). The addition of antibodies against HIF-1 or
ARNT to the binding reaction led to the formation of supershifted
complexes, the specificity of which was confirmed when the preimmune
serum was used. In conclusion, these results demonstrate that the
HRE-2 motif was recognized by the HIF-1-ARNT complex, in excellent agreement with the ability of the HRE-2 motif to mediate
hypoxia-inducible promoter activation in functional assays.
In the present report we have characterized the mechanism of induction of PAI-1 mRNA and protein expression by hypoxia. We observed that substantial up-regulation of PAI-1 mRNA and protein levels in human liver cells occurs mainly at oxygen concentrations below 2%. Interestingly, the PAI-1 gene was transcriptionally activated shortly after the onset of hypoxia, but the protein synthesis lagged behind the transcriptional up-regulation by several hours. Thus, our data demonstrate high responsiveness of PAI-1 in vitro in a narrow range of oxygen tensions and are in line with the observations in vivo in which the pathophysiologically low oxygen partial tensions found in tumors, during wound healing, and in the ischemic heart disease are accompanied by increased PAI-1 gene expression levels.26-28 To elucidate the molecular mechanisms involved in the hypoxic
induction, we analyzed the upstream promoter region of the
PAI-I gene. We have identified a functional
hypoxia-responsive element, HRE-2, located at positions With regard to the human HRE-2 motif, our analysis revealed that this 8 base-long recognition motif 5'-CACGTACA-3' was identical with the corresponding site present in the rat PAI-I promoter. Yet, the strong hypoxia-dependent induction of transcription and binding of HIF-1 that was observed with human HRE-2 motif is in striking contrast with the only weak hypoxic responses that were described in rat.24 In this context, it is noteworthy that there is adjacent, downstream to the human HRE-2, a 5'-CACAG-3' motif that matches a functionally essential sequence found downstream of the HIF-1-binding site in the human erythropoietin promoter.29 This motif, however, is absent from the rat promoter. Thus, the features associated specifically with the sequence immediately downstream from these particular HREs may provide a basis for the discrepancies in functional properties of the 2 elements from the 2 species. The HRE-4 and -5 sites were found to bind protein complexes independently of the oxygen partial tension. Consequently, the participation of HIF-1 in formation of these complexes can be excluded. However, each of the sites contained an E box sequence, which has been shown to bind a number of basic helix-loop-helix transcription factors,30 including the ARNT homodimer,31 or the Clock-BMAL heterodimer, that is involved in the control of the circadian expression of the PAI-1 gene.32 Given the fact that the levels of PAI-1 peak in early morning,33 concomitantly with low levels of oxygen in the circulation,34 it is plausible that HIF-1 is also involved in the control of the circadian oscillations of PAI-1. Further studies are thus required to elucidate the relative contributions of CLOCK and HIF-1 to this phenomenon.
We thank Professor P. Andreasen, University of Aarhus, Denmark, for the kind gift of the pEMBL8cat plasmid. The technical assistance of Lisbeth Rasmussen, Mette Bøgh, and Hanne Møller is highly appreciated.
Submitted September 26, 2001; accepted November 13, 2001.
Supported by Danish Medical Research council grant no. 9802548 and Danish Cancer Society grant no. 9821560.
P.E. is co-owner of the company that produced the prototype combined workbench-incubator sold to the host institute and used in the present work.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Trine Fink, Danish Cancer Society, Department of Virus and Cancer, Gustav Wieds Vej 10, 8000 Århus C, Denmark; e-mail: trine{at}virus.au.dk.
1. Booth NA. Fibrinolysis and thrombosis. Baillieres Best Pract Res Clin Haematol. 1999;12:423-433[Medline] [Order article via Infotrieve]. 2. Takazoe K, Ogawa H, Yasue H, et al. Increased plasminogen activator inhibitor activity and diabetes predict subsequent coronary events in patients with angina pectoris. Ann Med. 2001;33:206-212[Medline] [Order article via Infotrieve].
3.
Segarra A, Chacon P, Martinez-Eyarre C, et al.
Circulating levels of plasminogen activator inhibitor type-1, tissue plasminogen activator, and thrombomodulin in hemodialysis patients: biochemical correlations and role as independent predictors of coronary artery stenosis.
J Am Soc Nephrol.
2001;12:1255-1263 4. Hamsten A, de Faire U, Walldius G, et al. Plasminogen activator inhibitor in plasma: risk factor for recurrent myocardial infarction. Lancet. 1987;2:3-9[CrossRef][Medline] [Order article via Infotrieve].
5.
Harbeck N, Kruger A, Sinz S, et al.
Clinical relevance of the plasminogen activator inhibitor type1 6. Busso N, Nicodeme E, Chesne C, et al. Urokinase and type I plasminogen activator inhibitor production by normal human hepatocytes: modulation by inflammatory agents. Hepatology. 1994;20:186-190[CrossRef][Medline] [Order article via Infotrieve]. 7. Wohn KD, Schmidt T, Kanse SM, et al. The role of plasminogen activator inhibitor-1 as inhibitor of platelet and megakaryoblastic cell adhesion. Br J Haematol. 1999;104:901-908[CrossRef][Medline] [Order article via Infotrieve].
8.
Sironi L, Calvio AM, Arnaboldi L, et al.
Effect of valsartan on angiotensin II-induced plasminogen activator inhibitor-1 biosynthesis in arterial smooth muscle cells.
Hypertension.
2001;37:961-966 9. Underwood PA, Bean PA, Cubeddu L. Human endothelial cells grow poorly on vitronectin: role of PAI-1. J Cell Biochem. 2001;82:98-109[CrossRef][Medline] [Order article via Infotrieve].
10.
Nordt TK, Sawa H, Fujii S, Sobel BE.
Induction of plasminogen activator inhibitor type-1 (PAI-1) by proinsulin and insulin in vivo.
Circulation.
1995;91:764-770 11. Knudsen H, Olesen T, Riccio A, et al. 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. 1994;220:63-74[Medline] [Order article via Infotrieve]. 12. Tran-Thang C, Kruithof E, Lahm H, et al. Modulation of the plasminogen activation system by inflammatory cytokines in human colon carcinoma cells. Br J Cancer. 1996;74:846-852[Medline] [Order article via Infotrieve].
13.
Sandberg T, Eriksson P, Gustavsson B, Casslen B.
Differential regulation of the plasminogen activator inhibitor-1 (PAI-1) gene expression by growth factors and progesterone in human endometrial stromal cells.
Mol Hum Reprod.
1997;3:781-787 14. Fitzpatrick TE, Graham CH. Stimulation of plasminogen activator inhibitor-1 expression in immortalized human trophoblast cells cultured under low levels of oxygen. Exp Cell Res. 1998;245:155-162[CrossRef][Medline] [Order article via Infotrieve].
15.
Arts J, Grimbergen J, Toet K, Kooistra T.
On the role of c-Jun in the induction of PAI-1 gene expression by phorbol ester, serum, and IL-1alpha in HepG2 cells.
Arterioscler Thromb Vasc Biol.
1999;19:39-46
16.
Hua X, Miller ZA, Benchabane H, Wrana JL, Lodish HF.
Synergism between transcription factors TFE3 and Smad3 in transforming growth factor-beta-induced transcription of the Smad7 gene.
J Biol Chem.
2000;275:33205-33208
17.
Uchiyama T, Kurabayashi M, Ohyama Y, et al.
Hypoxia induces transcription of the plasminogen activator inhibitor-1 gene through genistein-sensitive tyrosine kinase pathways in vascular endothelial cells.
Arterioscler Thromb Vasc Biol.
2000;20:1155-1161
18.
Andrew AS, Klei LR, Barchowsky A.
Nickel requires hypoxia-inducible factor-1alpha, not redox signaling, to induce plasminogen activator inhibitor-1.
Am J Physiol Lung Cell Mol Physiol.
2001;281:L607-L615 19. Fink T, Ebbesen P, Zachar V. Quantitative gene expression profiles of human liver-derived cell lines exposed to moderate hypoxia. Cell Physiol Biochem. 2001;11:105-114[Medline] [Order article via Infotrieve]. 20. Villadsen JA, Mosborg Petersen P, Ebbesen P. Triple enclosure safety glove box with adjustable gas composition. Proceedings of the 15th ICCCS International Symposium Copenhagen: Denmark.; 2000:1-7. 21. Gradin K, McGuire J, Wenger RH, et al. Functional interference between hypoxia and dioxin signal transduction pathways: competition for recruitment of the Arnt transcription factor. Mol Cell Biol. 1996;16:5221-5231[Abstract].
22.
Whitelaw M, Pongratz I, Wilhelmsson A, Gustafsson JA, Poellinger L.
Ligand-dependent recruitment of the Arnt coregulator determines DNA recognition by the dioxin receptor.
Mol Cell Biol.
1993;13:2504-2514 23. Kvietikova I, Wenger RH, Marti HH, Gassmann M. The hypoxia-inducible factor-1 DNA recognition site is cAMP-responsive. Kidney Int. 1997;51:564-566[Medline] [Order article via Infotrieve].
24.
Kietzmann T, Roth U, Jungermann K.
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.
Blood.
1999;94:4177-4185
25.
Samoylenko A, Roth U, Jungermann K, Kietzmann T.
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.
2001;97:2657-2666
26.
Koong AC, Denko NC, Hudson KM, et al.
Candidate genes for the hypoxic tumor phenotype.
Cancer Res.
2000;60:883-887 27. Romer J, Lund LR, Eriksen J, et al. Differential expression of urokinase-type plasminogen activator and its type-1 inhibitor during healing of mouse skin wounds. J Invest Dermatol. 1991;97:803-811[CrossRef][Medline] [Order article via Infotrieve]. 28. Padro T, Steins M, Li CX, et al. Comparative analysis of plasminogen activator inhibitor-1 expression in different types of atherosclerotic lesions in coronary arteries from human heart explants. Cardiovasc Res. 1997;36:28-36[CrossRef][Medline] [Order article via Infotrieve].
29.
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.
1992;12:5447-5454 30. Murre C, McCaw PS, Baltimore D. A new DNA binding and dimerization motif in immunoglobulin enhancer binding, daughterless, MyoD, and myc proteins. Cell. 1989;56:777-783[CrossRef][Medline] [Order article via Infotrieve].
31.
Antonsson C, Arulampalam V, Whitelaw ML, Pettersson S, Poellinger L.
Constitutive function of the basic helix-loop-helix/PAS factor Arnt: regulation of target promoters via the E box motif.
J Biol Chem.
1995;270:13968-13972
32.
Maemura K, de la Monte SM, Chin MT, et al.
CLIF, a novel cycle-like factor, regulates the circadian oscillation of plasminogen activator inhibitor-1 gene expression.
J Biol Chem.
2000;275:36847-36851
33.
Sayer JW, Gutteridge C, Syndercombe-Court D, Wilkinson P, Timmis AD.
Circadian activity of the endogenous fibrinolytic system in stable coronary artery disease: effects of beta-adrenoreceptor blockers and angiotensin-converting enzyme inhibitors.
J Am Coll Cardiol.
1998;32:1962-1968 34. Smolensky MH, Portaluppi F. Chronopharmacology and chronotherapy of cardiovascular medications: relevance to prevention and treatment of coronary heart disease. Am Heart J. 1999;137:S14-S24[CrossRef][Medline] [Order article via Infotrieve].
© 2002 by The American Society of Hematology.
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  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]
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 Q. Cai, K. Lan, S. C. Verma, H. Si, D. Lin, and E. S. Robertson Kaposi's Sarcoma-Associated Herpesvirus Latent Protein LANA Interacts with HIF-1{alpha} To Upregulate RTA Expression during Hypoxia: Latency Control under Low Oxygen Conditions. J. Virol., August 1, 2006; 80(16): 7965 - 7975. [Abstract] [Full Text] [PDF]
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 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]
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 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]
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Top
Abstract
Introduction
,
a multifaceted proteolytic factor.
Onkologie.
2001;24:238-244