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Prepublished online as a Blood First Edition Paper on September 12, 2002; DOI 10.1182/blood-2002-06-1693.
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Institut für Biochemie und Molekulare
Zellbiologie, Göttingen, Germany.
The expression of the plasminogen activator inhibitor-1
(PAI-1) gene is enhanced by insulin both in vivo
and in various cell types. Because insulin exerts a number of its
biologic activities via the phosphatidylinositol 3-kinase and protein
kinase B (PI3K/PKB) signaling pathway, it was the aim of the present
study to investigate the role of the PI3K/PKB pathway in the expression
of the PAI-1 gene and to identify the insulin responsive
promoter sequences. It was shown that the induction of PAI-1 mRNA and
protein expression by insulin and mild hypoxia could be repressed by
the PI3K inhibitor wortmannin. Overexpression of a constitutively
active PKB led to induction of PAI-1 mRNA expression and of luciferase
(Luc) activity from a gene construct containing 766 bp of the rat PAI-1 promoter. Mutation of the hypoxia response elements (HRE-1 and HRE-2)
in rat PAI-1 promoter, which could bind hypoxia inducible factor-1
(HIF-1), abolished the induction of PAI-1 by insulin and PKB.
Insulin and the constitutive active PKB also induced Luc expression in
cells transfected with the pGl3EPO-HRE Luc construct, containing 3 copies of the HRE from the erythropoietin gene in front of the SV40
promoter. Furthermore, insulin and the active PKB enhanced all 3 HIF
The broad-spectrum serine protease plasmin is
activated by the proteases, tissue-type (tPA) and urokinase-type (uPA)
plasminogen activators.1 The tPA and uPA activity is
regulated, in part, by plasminogen activator inhibitors (PAIs) that are
glycoproteins of the serine protease inhibitor (serpin)
superfamily.2 Among 2 identified inhibitors, PAI-1 and
PAI-2, PAI-1 is the primary physiologic inhibitor of both tPA and uPA.
It can be produced by platelets, vascular endothelial
cells,3 vascular smooth muscle cells,4 and
several nonvascular cell types, among them hepatocytes.5,6
PAI-1 regulates fibrinolysis in many normal and pathologic conditions
such as atherosclerosis, coronary heart disease, wound healing, and
cancer metastasis.7-9 It was shown that hyperinsulinemia especially associated with obesity, hypertension, and diabetes type 2 could increase PAI-1 levels in blood.10-13 Furthermore, insulin induced PAI-1 expression in vitro in various cell types, including primary human hepatocytes,14,15 HepG2
cells,16,17 and arterial endothelial
cells.18,19
Insulin signaling involves second messengers, including members of the
phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein
kinase (MAPK) cascades.20 The PI3K, which generates phosphatidylinositol-3,4,5-phosphate (PI3,4,5P3), has a key
role in the metabolic actions of insulin.21
PI3,4,5P3 regulates the activity or subcellular
localization of a variety of signaling molecules such as
phosphatidylinositol-dependent kinase (PDK) and protein kinase B (PKB)
known as Akt, which are also involved in the transmission of the
insulin signal.22,23
The expression of the PAI-1 gene and many other genes,
expression of which can be induced by insulin, such as genes for the glucose transporters,24 several glycolytic
enzymes,25 nitric oxide (NO)
synthase,26 erythropoietin (EPO),27 and
vascular endothelial growth factor (VEGF),28 can also be
induced by hypoxia.29-33 The rat PAI-1
gene was induced by hypoxia via an O2 responsive promoter
sequence ( It was shown that PAI-1 gene expression was induced by both
insulin and mild hypoxia. Expression of a constitutively activated PKB
enhanced PAI-1 expression and PAI-1 promoter-dependent luciferase (Luc)
activity. Concomitantly, insulin and PKB enhanced HIF-1 All biochemicals and enzymes were of analytical grade and were
purchased from commercial suppliers.
Animals
Cell culture experiments
Plasmid constructs The pGl3PAI-766 plasmid, containing the rat PAI-1 promoter 5'-flanking region41 from 766 to +31, as well as
pGl3PAI-766M1 and pGl3PAI-766M2, was described before.34
The luciferase gene construct pGl3EPO-HRE containing 3 HREs from the
EPO gene in front of the SV40 promoter was
described.42 The vectors expressing the constitutively
active form of PKB (myrPKB) and a dominant-negative PKB (PKB-K179A)
were a kind gift from Dr D. Stokoe and have been already
described.43
RNA preparation and Northern analysis Isolation of total RNA and Northern analysis were performed as described.34 Digoxigenin (DIG)-labeled antisense RNAs served as hybridization probes; they were generated by in vitro transcription from pBS-PAI-1, pBS-rHIF2 -650, and pBS-GK using T3 RNA
polymerase or from pCRII-rHIF1-800 and
pCRIITOPO-rHIF3-200042 and pBS- actin using T7 RNA
polymerase and RNA labeling mixture containing 3.5 mM 11-DIG-uridine
triphosphate (UTP), 6.5 mM UTP, 10 mM guanosine triphosphate
(GTP), 10 mM cytosine triphosphate (CTP), and 10 mM adenosine
triphosphate (ATP). The use of a full-length HIF-3 probe was
appropriate because liver and HepG2 cells appeared not to express the
HIF-3 splice variant IPAS.44 Hybridizations and
detections were carried out essentially as described
before.34 Blots were quantified with a videodensitometer
(Biotech Fischer, Reiskirchen, Germany).
Western blot analysis PAI-1, HIF-1, HIF-2, HIF-3, PKB, and phosphoPKB
(PKB-S473) Western blot analysis was carried out as
described.45-47 In brief, media or lysates from primary
cultured hepatocytes were collected, and the protein content was
determined by using the Bradford method. Protein (50 µg) 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, Heidelberg,
Germany) was used in a 1:200 dilution. The secondary antibody
was a goat antirabbit immunoglobulin G (IgG; Santa Cruz Biotechnology,
Santa Cruz, CA) and used in a 1:2000 dilution. The primary antibodies
against HIF-1, HIF-2, and HIF-3 were described
earlier42 and used in a 1:800 dilution. The primary rabbit antibody against Golgi membrane (GM; Bioscience,
Göttingen, Germany) was used in a 1:8000 dilution. The secondary
antibody was a goat antirabbit IgG horseradish peroxidase (HRP;
Santa Cruz Biotechnology), used in a 1:2000 dilution. The PKB and
PKB-S473 antibody (Cell Signaling, Frankfurt, Germany) was used in a
1:1000 dilution; the secondary antibody was an antirabbit IgG and used as above. The monoclonal mouse anti-EE antibody (Hiss Diagnostics, Freiburg, Germany) was used in a 1:1000 dilution; the secondary antibody was an antimouse IgG HRP and used as above. The enhanced chemiluminescence (ECL) Western blotting system (Amersham) was used for detection. Under these conditions PAI-1 was seen as a double
band, the major 49-kDa band and the minor 46-kDa band,45 HIF-1 was visible as a 120-kDa band, HIF-2 as a 130-kDa band, and
HIF-3 as a 74-kDa band.42 PKB was visible as a 60-kDa
band.48
Cell transfection and luciferase assay Freshly isolated rat hepatocytes (about 1 × 106 cells per dish) were transfected as described,46 thereby controlling transfection efficiency by cotransfection of 0.25 µg Renilla Luciferase expression vector (pRLSV40) (Promega). Additionally, 2 µg of the appropriate PAI-1 or EPO-HRE promoter Firefly luciferase construct was transfected together with 500 ng PKB expression vectors or in the controls with 500 ng empty control vector. For Northern blot and Western blot experiments cells were transfected with 2 µg PKB-K179A expression vector, myrPKB expression vector, or control vector. After 5 hours the medium was changed, and the cells were cultured under normoxia for 19 hours. Then, medium was changed again, and the cells were further cultured for 24 hours under normoxia or mild hypoxia. Three hours before harvesting the cells were treated with insulin (10 nM).Preparation of nuclear extracts and electrophoretic mobility shift assay (EMSA) Nuclear extracts were prepared by modification of a standard protocol essentially as described.34,49 The sequence of the PAI-1 oligonucleotide used for the EMSA is 5'-TCTCACACACGTACACACACGTGTC-3' (182/157). Equal amounts of
complementary oligonucleotides were annealed and labeled by 5'-end
labeling with  -32P]ATP (Amersham) and T4 polynucleotide
kinase (MBI). They were purified with the Nucleotide Removal Kit
(Qiagen). Binding reactions were carried out in a total volume of 20 µL containing 50 mM KCl, 1 mM MgCl2, 1 mM EDTA
(ethylenediaminetetraacetic acid), 5% glycerol, 10 µg nuclear
extract, 250 ng poly d(I-C), and 5 mM dithioerythrol (DTE).
After preincubation for 5 minutes at room temperature, 1 µL labeled
probe (104 cpm) was added, and the incubation was continued
for an additional 10 minutes. For supershift analysis 1 µL HIF-1
antibody was added to the EMSA reaction that was then incubated at
4°C for 2 hours. The electrophoresis was then performed with a 5%
nondenaturing polyacrylamide gel in TBE buffer (89 mM Tris
(tris(hydroxymethyl)aminomethane), 89 mM boric acid, 5 mM EDTA) at
200 V. After electrophoresis the gels were dried and exposed to a
phosphorimager screen.
Induction of PAI-1 expression by insulin via the PI3K pathway Treatment of cells with different concentrations of insulin led to a concentration-dependent enhancement of PAI-1 mRNA levels. In the range of physiologic insulin concentrations up to 10 nM, PAI-1 mRNA was induced by about 3-fold under normoxia. The insulin concentration of 100 nM did not give rise to a more dramatic PAI-1 mRNA induction (Figure 1A). Under hypoxia the insulin-dependent PAI-1 mRNA induction started at 1 nM insulin, and then the induction was more pronounced than under normoxia. Maximal activation by about 7-fold under hypoxia was achieved with an insulin concentration of 100 nM. For further experiments an insulin concentration of 10 nM was chosen (Figure 1A). Treatment of hepatocytes with insulin enhanced PAI-1 mRNA levels to a transient maximum after 3 hours under both normoxia and hypoxia. PAI-1 mRNA levels then decreased again. Thus, for the experiments the cells were treated with insulin for 3 hours (data not shown).
In cultured hepatocytes hypoxia induced PAI-1 mRNA and protein by about 5-fold in line with a previous study.34 Treatment of cells with insulin enhanced PAI-1 mRNA and protein by about 3-fold under normoxia and by about 6-fold under hypoxia. The PI3K inhibitor wortmannin did not affect PAI-1 mRNA and protein expression under normoxia but reduced the PAI-1 mRNA and protein induction under hypoxia to about 2.2-fold compared with the controls under normoxia. The presence of both insulin and wortmannin completely abolished PAI-1 mRNA and protein induction under both normoxia and hypoxia (Figure 1B). To control the action of insulin the insulin-dependent glucokinase (GK) mRNA induction was measured. GK mRNA was hardly detectable in the controls, ie, without insulin. Treatment of the cells with insulin enhanced GK mRNA under normoxia and was more pronounced under hypoxia that was in line with previous findings.34 Again, wortmannin abolished the induction of GK mRNA by insulin (Figure 1C). Thus, these findings indicate that the activation of PAI-1 expression by insulin is mediated via the PI3K pathway. Induction of PAI-1 mRNA expression by PKB A further downstream-acting component in the PI3K pathway is PKB, also known as Akt. To investigate the involvement of PKB in the pathway contributing to the enhanced levels of PAI-1, cells were transfected with expression vectors encoding either a dominant-negative protein kinase B (PKB-K179A) or a constitutively active myristoylated variant of PKB (myrPKB). Similar to wortmannin transfection of the PKB-K179A expression vector resulted in decreased PAI-1 mRNA levels under hypoxia when compared with the control. The transfection of the vector for the constitutively active myrPKB enhanced PAI-1 mRNA levels by about 2-fold under normoxia and by about 4-fold under hypoxia. Thus, these results indicate that also the PKB pathway can contribute to enhanced PAI-1 levels (Figure 2).
Insulin-dependent activation of PAI-1 promoter Luc gene constructs To investigate whether the insulin-dependent induction of the PAI-1 gene is mediated by a distinct promoter sequence, primary hepatocytes were transfected with Luc gene constructs driven by the hypoxia-inducible rat PAI-1 gene promoter. In pGl3PAI-766 Luc-transfected cells, Luc activity was enhanced by about 3-fold under hypoxia in line with previous studies.34,49 Treatment of the transfected cells with insulin enhanced Luc activity by about 2-fold under normoxia and by about 5-fold under hypoxia (Figure 3).
Because it was shown that the transcription factor HIF-1 binding to HREs can be a target in the PKB signaling pathway, it was further tested whether the mutation of the HREs in the PAI-1 promoter abolished the insulin-dependent Luc induction. Transfection of the PAI-1 promoter Luc construct in which the HRE-1 was mutated (pGl3PAI-766M1) abolished the induction of Luc activity by hypoxia as well as the insulin-dependent induction under both normoxia and hypoxia. Similarly, mutation of the HRE-2 site in pGl3PAI-766M2 also attenuated the induction of Luc activity by hypoxia in line with a previous study.34 Treatment of the transfected cells with insulin did not increase Luc activity as well (Figure 3). This finding indicated that indeed HIF-1 was involved in the insulin-dependent activation of the PAI-1 gene. To further substantiate this finding, the cells were transfected with a Luc gene construct containing 3 isolated HREs from the EPO gene in front of the SV40 promoter. Transfection of the pGl3EPO-HRE Luc construct resulted in higher Luc activity by about 3-fold when the cells were exposed to hypoxia. Treatment of the pGl3EPO-HRE Luc-transfected cells with insulin enhanced Luc activity by about 2-fold under normoxia and by about 4-fold under hypoxia (Figure 3). The involvement of PKB was tested by cotransfection of the PAI-1 promoter Luc constructs with the expression vector encoding the constitutively activated PKB (myrPKB). Cotransfection of the wild-type PAI-1 promoter with the myrPKB expression vector elicited enhanced Luc activity by about 5-fold under normoxia and by about 8-fold under hypoxia. The mutation of either HRE site, HRE-1 or HRE-2, completely abolished the myrPKB-mediated induction of Luc activity. The cotransfection of the pGl3EPO-HRE Luc construct with the myrPKB expression vector enhanced Luc activity by about 3-fold under normoxia and by about 6-fold under hypoxia (Figure 3). Thus, it appears that the PAI-1 gene activation by insulin is mediated via the PI3K/PKB/HIF-1 pathway. Enhancement of HIF ,
HIF-2, or HIF-3, Northern and Western blot analyses were
performed. Treatment of hepatocytes with insulin under normoxia and
hypoxia did not enhance the mRNA levels of the 3 HIF -subunits
(Figure 4A). In contrast, the HIF-1
and HIF-2 protein levels were increased by about 4-fold and that of
HIF-3 by about 2-fold under hypoxia. Insulin treatment for 3 hours
mediated about a 3-fold enhancement of all HIF- protein levels under
normoxia. Under hypoxia insulin treatment increased the HIF-1 levels
by about 6-fold, the HIF-2 levels by about 5-fold, and the HIF-3
levels by about 3-fold compared with the normoxic control (Figure
4B,C). The insulin-dependent increase of the HIF -subunit protein
levels was mediated via PI3K because the PI3K inhibitor wortmannin
abolished the insulin-dependent enhancement of the HIF -subunit
proteins (Figure 4C).
Similar to insulin, transfection of the cells with the vector for the
constitutively active PKB enhanced the HIF-1 Insulin-mediated enhanced binding of HIF-1 to the hypoxia-responsive sequence of the PAI-1 promoter To verify that the insulin-dependent and PKB-mediated induction of PAI-1 promoter controlled Luc activity and the accumulation of the HIF-1 protein is reflected by enhanced binding of HIF-1 to the PAI-1
HREs, EMSAs with nuclear extracts from cells cultured under either
normoxia or hypoxia and transfected with the vector for the
constitutively active PKB were performed. After incubation of the PAI-1
HRE1/2 oligonucleotide with nuclear extracts from cells cultured under
normoxia, a DNA-protein complex could be detected, the formation of
which was enhanced when nuclear extracts prepared from cells cultured
under hypoxia were used (Figure 5). Usage
of the nuclear extracts prepared from the myrPKB-transfected cells
cultured under both normoxia and hypoxia also displayed the enhanced
formation of the DNA-protein complex as observed with nuclear extracts
from cells cultured under hypoxia. To confirm the presence of the
HIF-1 protein in the observed complex, an anti-HIF-1 antibody
was included in the binding reaction. When nuclear extracts from the
cells transfected with myrPKB were treated with the anti-HIF-1
antibody, a supershifted complex was observed (Figure 5). These
findings further substantiate the conclusion that PKB acts via HIF-1
and the HRE sites of the PAI-1 promoter.
The present study has shown that the insulin-dependent activation of the rat PAI-1 gene expression is mediated via the PI3K/PKB pathway and the transcription factor HIF-1 binding to the HRE sites in the PAI-1 promoter. Oxygen-independent activation of HIF-1-regulated genes Hypoxic HIF-1 activation stimulates its binding to HREs, subsequently leading to the induction of genes whose products either increase availability of oxygen, such as EPO and VEGF, or promote metabolic adaptation to oxygen deprivation, such as glucose transporters or glycolytic enzymes.52 It is known that genes responsible for glucose and energy metabolism, as well as EPO and VEGF, are induced also by insulin which indicates that hypoxia and insulin signaling pathways could be interrelated.53,54 The results of our study confirm previous observations that exposure of human embryonic kidney 293 cells to insulin38 and HepG2 hepatoma cells to insulin39 resulted in the induction of HIF-1 protein levels. EMSA experiments with nuclear extracts from
HepG2 cells and L8 rat skeletal muscle myoblasts, cultured under
normoxia and treated with insulin, and an oligonucleotide corresponding
to the HRE sequence of the EPO gene showed the formation of
the active HIF-1 complex.37 Furthermore, insulin induced
the expression of a Luc reporter construct containing 5 copies of the
EPO-HRE as enhancer37 that is also in line with the present
findings. Moreover, our study for the first time showed that primary
hepatocytes treated with insulin and cultured under mild hypoxia
contained higher levels of the HIF-1 protein and higher Luc
activities from the PAI-1 promoter and the EPO-HRE Luc reporter
construct when compared with the insulin-untreated hepatocytes cultured
under hypoxia. This finding also indicates that insulin treatment and
exposure of the cells to hypoxia have an additive effect on the
HIF-1 activity.
Beside insulin, other hormones, growth factors, and clotting factors
such as insulinlike growth factor (IGF),37,38
angiotensin II,55 platelet-derived growth factor
(PDGF),55 thrombin,56 and tumor necrosis
factor Signaling pathways involved in the activation of HIF-1 The mechanisms by which hypoxia stimulates activation of HIF-1 are not understood to the last detail. Proposed models involve signal transduction pathways via oxygen-binding hemoproteins and generation of reactive oxygen species,52 as well as via oxygen-dependent prolyl hydroxylation58,59 and asparaginyl hydroxylation.60 The latter mechanisms have been shown to be of major importance for HIF-subunit protein stability and
coactivator recruitment. Under normoxia HIF-1 and HIF-2
destabilization is conferred by the O2-dependent
hydroxylation of at least 2 proline residues within the
O2-dependent degradation domains, enabling the binding of
the von Hippel-Lindau tumor suppressor protein (pVHL), a component of
an E3 ubiquitin ligase complex that targets the HIF -subunits for
degradation by the ubiquitin-proteasome pathway.61 In a similar manner the hydroxylation of an asparaginyl residue in the
C-terminal transactivation domain of HIF-1 and HIF-2 prevents the
recruitment of the coactivator CBP/p300, thus reducing the HIF-1 and
HIF-2 transactivation potential.60 Furthermore, it was
suggested that MAP kinases modulate the transcriptional activity of
HIF-1 via phosphorylation of HIF-1 at its regulatory domain.62,63 In addition, the PI3K/PKB pathway was
proposed to regulate the activity of HIF-1 mainly via stabilization of the HIF-1 protein.63,64 In line with this the present
study and a study with HepG2 cells demonstrated that insulin induced HIF-1 accumulation, HIF-1 DNA-binding, and PAI-1, VEGF, and EPO synthesis independently from MAP kinases via the PI3K
pathway.39 Furthermore, under hypoxia the PI3K/PKB pathway
was responsible for the induction of VEGF expression in Ras-transformed
NIH3T3 cells65 and inactivation of the PKB target
glycogen synthetase kinase 3 (GSK-3) in HT1080
cells.66 The PI3K/PKB pathway was also shown to be
involved in the NO-dependent,67
thrombin-dependent,56 heregulin-dependent68
and, as in the present and another recent study,69 the
insulin-dependent activation of HIF-1 under normoxia. Thereby, insulin
appeared to act via a translational-dependent pathway because it was
currently shown that cycloheximide inhibited the insulin-dependent
enhancement of the HIF-1 protein levels69 and, as in
this study, did not affect the HIF-1 mRNA expression69 (Figure 4A).
In our study the insulin-dependent accumulation of all 3 HIF
In summary, the findings of this study with the PAI-1 gene are another example for the role of the PI3K/PKB pathway as a key regulatory cascade responsible for insulin signaling that may undergo a cross talk with the hypoxia-dependent signaling pathway in a cell type-specific manner. PI3K/PKB pathway in the activation of the PAI-1 gene expression The involvement of HIF-1 and the PI3K/PKB pathway in the regulation of PAI-1 gene expression by insulin has not been shown previously. However, the proposal that the PI3K may have an effect on the stimulation of PAI-1 expression came from the observations that nerve growth factor-induced PAI-1 mRNA expression was inhibited by wortmannin in rat pheochromocytoma PC12 cells73 and that the insulin-induced human PAI-1 protein secretion was inhibited by LY294002 in HepG2 cells.74 These results concur with our study. But in contrast to our experiments, the only other study, in which the signal transduction pathway leading to the PAI-1 induction by insulin was investigated, showed that downstream from PI3K protein kinase C (PKC) and MAP kinases were activated.74 Thus, it seems likely that at least in human cells downstream from PI3K more than one pathway is involved in the regulation of PAI-1 gene expression by insulin.In contrast to our study, the transcription factor responsible for
activation of human PAI-1 expression via the PKC/MAPK pathway in HepG2
cells as well as the exact insulin responsive element in the human
PAI-1 promoter has not been identified. However, it was demonstrated by
cotransfection and EMSA experiments that 3 regions of the human PAI-1
promoter, Insulin- and HIF-dependent PAI-1 gene expression under physiologic and pathophysiologic conditions Induction of PAI-1 expression by insulin via HIF-1 might have importance for the development of cardiovascular disease. HIF-1 has been implicated to be involved in neovascularization of ischemic myocardium via activation of VEGF expression.33,76 The levels of plasma PAI-1 are elevated in hyperinsulinemia, hypertriglyceridemia, hypertension, obesity, and diabetes type 2 that are all characteristic for the insulin resistance syndrome associated with a highly increased risk of cardiovascular disease.77,78 Impaired fibrinolysis because of overexpression of PAI-1 has been implicated as one of the mechanisms responsible for coronary artery disease under hyperinsulinemia.79 However, data concerning the up-regulation of human PAI-1 by insulin in vivo are somewhat contradictory. Some in vivo studies investigating the effects of insulin infusion found no effect on the levels of PAI-1 in blood or even a decrease of PAI-1 levels and activity.80-82 However, induction of PAI-1 by insulin was found when the perfused forearm model was used to study the local effects of insulin infusion.13 Furthermore, in combination with hypertriglyceridemia and hyperglycemia, hyperinsulinemia was shown to increase PAI-1 plasma levels.83 Thus, further investigations are still necessary to elucidate the complete physiologic role of PAI-1 activation by insulin and its molecular mechanisms.
We thank Dr D. Stokoe (Cancer Research Institute, University of California, San Francisco) for the kind gift of the myrPKB and PKB-K179A plasmids and Dr T. D. Gelehrter (Department of Human Genetics, University of Michigan Medical School, Ann Arbor) for the kind gift of PAI-1 cDNA.
Submitted June 10, 2002; accepted August 26, 2002.
Prepublished online as Blood First Edition Paper, September 12, 2002; DOI 10.1182/blood-2002-06-1693.
Supported by the Deutsche Forschungsgemeinschaft SFB 402, Teilprojekt A1, and GRK 335.
Kurt Jungermann died on May 10, 2002.
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: Thomas Kietzmann, Institut für Biochemie und Molekulare Zellbiologie, Humboldtallee 23, D-37073 Göttingen, Germany; e-mail: tkietzm{at}gwdg.de.
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Abstract
Introduction
.05.
(C) Representative Northern and Western blot. For Northern analysis 15 µg total RNA was hybridized to digoxigenin-labeled PAI-1, GK, and
