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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Institut für Biochemie und Molekulare
Zellbiologie, Humboldtallee 23, Göttingen, Germany.
Plasminogen activator inhibitor-1 (PAI-1) expression is induced by
hypoxia (8% O2) via the PAI-1 promoter region The tissue-type and the urokinase-type plasminogen
activators (tPA and uPA) are serine proteases converting the inactive
zymogen plasminogen to the active endopeptidase plasmin.1
The tPA and uPA activity is regulated, in part, by plasminogen
activator inhibitors (PAIs).2 Among 2 identified
inhibitors, PAI-1 and PAI-2, PAI-1 is the primary physiologic inhibitor
of both tPA and uPA.3 PAI-1 is a 50-kd glycoprotein from
the serpin superfamily.4 It can be produced by platelets,
vascular endothelial cells,5 vascular smooth muscle
cells,6 and several nonvascular cell types,7,8 including hepatocytes.9 PAI-1 has
also been identified as a component of the extracellular
matrix.10
The plasminogen activator inhibitors are involved in many functions,
both under normal and pathological conditions, including fibrinolysis,
extracellular matrix turnover, and fibrosis.11 PAI-1 also
participates in wound healing and cancer metastasis.12,13
Certain pathophysiologic processes in which PAI-1 levels increase (eg,
prethrombotic events, hemorrhage, and thrombus formation) are
associated with hypoxia. PAI-1 gene expression was induced by mild
hypoxia (8% O2) via an O2-responsive promoter
sequence ( The HRE-1 and also HRE-2 include a CACGTG-like sequence that can be
recognized by transcription factors containing basic helix-loop-helix (bHLH) domains. Besides HIF-1 consisting of the bHLH-PAS
(Per-ARNT-Sim) domain proteins HIF-1 Two different ubiquituously expressed forms of USF (USF-1 and USF-2)
with different molecular weights have been identified; however, their
relative abundance varies in different cell types.19,20 The USF family members are quite different in their N-terminal amino
acid sequences, whereas their DNA binding and dimerization domains were
highly identical.19 Thus, it may be likely that proteins
of the bHLH-zip family can bind to the PAI-1 HRE sites. Therefore, it
was the aim of the present study to identify the HRE-1 binding factor
and to investigate its role in the regulation of PAI-1 expression using
primary cultured rat hepatocytes as a model system. By using
electrophoretic mobility shift assays (EMSAs) and supershift assays, it
was shown that USF proteins bound to the hypoxia-responsive elements of
the rat PAI-1 gene promoter, mainly HRE-1, and that overexpression of
USF-2a inhibited PAI-1 expression under normoxia and hypoxia to the
same extent. Mutations of both HREs abolished the inhibition of PAI-1
with a more prominent effect on HRE-1. Furthermore, the inhibitory action of USF-2 at HRE-2 could be antagonized by transfection of a
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 region21 from 766 to +31, as well as
pGl3PAI-766M1 and pGl3PAI-766M2 were previously
described.14 Plasmids pGl3PAI-276, pGl3PAI-276M1, and
pGl3PAI-276M2 were constructed from pGl3PAI-766, pGl3PAI-766M1, and
pGl3PAI-766M2 by excision of a 490-base pair (bp) KpnI
fragment and subsequent relegation of the remaining vector. The
construct L-type pyruvate kinase-183 luciferase (PKL-183 Luc) was constructed by excision of the PKL-183 promoter
sequence from PKL-183 CAT,22 with
SacI and subsequent ligation into the SacI sites
of pGl3 basic (Promega, Heidelberg, Germany). The human USF-2a,
 HU2a, and TDU2 plasmids were a kind gift from Dr A. Kahn and Dr
M. Raymondjean and, as well as the HIF-1 expression vector, they
have been already described.14,23
RNA preparation and Northern analysis Isolation of total RNA and Northern analysis were performed as described.24 Digoxigenin (DIG)-labeled antisense RNAs served as hybridization probes; they were generated by in vitro transcription from pBS-PAI-1 and pBS-PKL using T3 RNA polymerase or from pBS- -actin using T7 RNA polymerase and RNA
labeling mixture containing 3.5 mM 11-DIG-UTP, 6.5 mM UTP, 10 mM GTP,
10 mM CTP, and 10 mM ATP. Hybridizations and detections were carried
out essentially as previously described.24 Blots were
quantified with a videodensitometer (Biotech Fischer, Reiskirchen, Germany).
Western blot analysis Western blot analysis was carried out as described.25 In brief, media from primary cultured hepatocytes were collected, and the protein content was determined using the Bradford method. A total of 50 µg protein was loaded onto a 10% sodium dodecyl sulfate-polyacrylamide gel and after electrophoresis blotted onto nitrocellulose membranes. The primary rabbit antibody against rat PAI-1 (American Diagnostics, Greenwich, CT) was used in a 1:200 dilution. The secondary antibody was a goat antirabbit immunoglobulin G (Santa Cruz Biotechnology, Santa Cruz, CA) used in a 1:2000 dilution. The PKL Western analysis was performed as described 26 except that the primary antibody was a mouse monoclonal antibody against rat PKL,27 which was used in a 1:100 dilution. The secondary antibody was an antimouse IgG horseradish peroxidase (Santa Cruz Biotechnology) used in a 1:2000 dilution. The enhanced chemiluminescence Western blotting system (Amersham, Freiburg, Germany) 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,25 and PKL was visible as a 60-kd band.Cell transfection and Luc assay Freshly isolated rat hepatocytes (about 1 × 106 cells per dish) were transfected as described.26 In brief, 2 µg of the appropriate PAI-1 or PKL promoter Firefly Luc construct was transfected together with 500 ng USF-2a,HU2a, or TDU2 expression vectors or in the controls with
500 ng USF-2a backbone plasmid pCMV. In competition experiments, 2 µg
of the appropriate PAI-1 promoter Luc construct was transfected
together with 500 ng USF-2a and 500 ng HIF-1 expression vector or
with 500 ng USF-2a or HIF-1 vector plus 500 ng pCMV vector. In the
controls, 1 µg pCMV vector was used with 2 µg of the appropriate
PAI-1 construct. 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.
Preparation of nuclear extracts and EMSA Nuclear extracts were prepared by modification of a standard protocol,28,29 with buffers A and C containing 0.5 mM dithioerythritol (Sigma, Deisenhofen, Germany), 0.4 mM phenylmethylsulfonyl fluoride (Serva), 2 µg leupeptin per milliliter (Roche, Mannheim, Germany), 2 µg pepstatin per milliliter (Roche), 2 µg aprotinin per milliliter (Bayer, Leverkusen, Germany), 1 mM sodium vanadate (Sigma), and the "complete" protease inhibitor cocktail tablets (Roche) essentially as described.14 The sequences of the PAI-1 oligonucleotides used for the EMSA are shown in Figure 1A. Equal amounts of complementary oligonucleotides were annealed and labeled by 5'-end labeling with [ -32P]ATP (Amersham) and T4 polynucleotide kinase
(MBI, St Leon-Rot, Germany). They were purified with the Nucleotide
Removal Kit (Qiagen, Hilden, Germany). Binding reactions were carried
out in a total volume of 20 µL containing 50 mM KCl, 1 mM
MgCl2, 1 mM ethylenediaminetetraacetic acid, 5% glycerol,
10 µg nuclear extract, 250 ng poly(dIdC), and 5 mM dithioerythritol.
For competition analyses, a 1-, 5-, 10-, or 50-fold molar excess of
unlabeled annealed oligonucleotide was added. 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 USF-1 (C20),
USF-2 (N18), Myc (C33), Max (C17), or SP-1 (PEP2-G) antibodies (Santa
Cruz Biotechnology) as well as a rabbit preimmune serum14
were added to the EMSA reaction and 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, 89 mM boric acid, 5 mM
ethylenediaminetetraacetic acid) at 200 V. After electrophoresis the
gels were dried and exposed to a phosphorimager screen.
Binding of USF-2a to HRE sequences of the rat PAI-1 promoter There are 2 E-box-like sequences, E1 (175/170) and E2
(165/160), inside the "C" site of the rat PAI-1 promoter, which was identified by footprint analysis with liver nuclear
extracts.30 Both E-boxes acted as HREs and were thus
designated HRE-1 and HRE-2. Besides HRE-2 (165/160), which was
found to bind the HIF-1, HRE-1 (175/170) bound a so far unknown
factor.14 Because the CACGTG motif, which includes the
canonical E-box sequence CANNTG, appears to be the common recognition
site for bHLH transcription factors such as USF,16 it was
supposed that the HRE-1 or even both HREs can be bound by USF. The
first potential USF binding element, HRE-1 5'-CACGTA-3', matches the
consensus sequence in 5 of 6 bp. The second potential USF binding
element, HRE-2 5'-CACGTG-3', contains the canonical USF binding
sequence CACGTG (Figure 1).
The binding of nuclear proteins to oligonucleotide probes spanning the
HRE-1 and HRE-2 sites of the rat PAI-1 promoter was examined by EMSA.
The oligonucleotide To investigate the presence of USF in these complexes, antibodies against USF-1 and USF-2 were included in the binding reaction. The USF-1 antibody was raised against a C-terminal 18-amino acid polypeptide of human origin, whereas the USF-2 antibody was raised against an N-terminal 18-amino acid peptide of mouse origin. Addition of the USF-1 antibody to the EMSA reaction strongly reduced, whereas the USF-2 antibody inhibited, the formation of the DNA complex with the HRE-1 oligonucleotide, and both led to a supershifted complex. With the HRE-2 oligonucleotide, the USF antibodies slightly produced a supershifted complex, which appeared stronger with the USF-2 antibody. Furthermore, it appeared that the addition of the USF antibodies favored binding of the HIF-1 complex to the HRE-2. To ensure specificity of the supershifts mediated by the USF antibody, the EMSA was also performed in the presence of a rabbit preimmune serum and an antibody against the GC-box binding factor SP-1. Neither the preimmune serum nor the SP-1 antibody influenced the complex formation with the HRE-1 and the HRE-2 oligonucleotides. To test whether other members of the bHLH family such as Myc or Max may participate in binding to the HRE-1 and HRE-2, supershifts with Myc and Max antibodies were performed. Addition of antibodies against Myc or Max did not result in a supershift or inhibition of complex formation (Figure 1B). Thus, it appears that USF-2 could play the more important role in PAI-1 gene expression. To investigate whether HRE-1 could bind the USF complex with higher affinity, competition experiments were performed. The formation of the USF complex with the HRE-1 oligonucleotide could be clearly reduced by unlabeled HRE-1 starting with a 5-fold molar excess of cold HRE-1 oligonucleotides (Figure 1C, upper part, left). At this point it appeared that only 2 faint complexes were visible, consistent with the finding that both USF-1 and USF-2 can bind to the HRE-1. In contrast, the competition of HRE-1 binding with the cold HRE-2 oligonucleotide was not as prominent as with the unlabeled HRE-1 oligonucleotide; clear reduction of binding activity was visible at a 10- to 50-fold molar excess of HRE-2 oligonucleotide (Figure 1C, upper part, right). Vice versa it could be shown by competition of the HRE-2 binding complexes that, again, unlabeled HRE-2 competed with higher affinity for the hypoxia-inducible as well as the constitutive complex than the HRE-1 (Figure 1C, lower part). These findings together indicated that the PAI-1 HRE sites could be bound by USF proteins and that the HRE-1 site is the major USF binding site. Inhibition of PAI-1 expression and induction of PKL expression by overexpression of USF-2a under both normoxia and mild hypoxia To investigate the role of USF-2a in the regulation of PAI-1 gene expression, primary cultured rat hepatocytes were transfected with expression vectors encoding wild-type human USF-2a or the mutant USF-2a proteinHU2a lacking the second helix of the HLH domain and thus
unable to bind DNA and to dimerize.23 Hepatocytes cultured
under mild hypoxia and transfected with the empty control vector
displayed an about 2-fold-enhanced PAI-1 messenger RNA (mRNA) level,
in line with a previous study.14 In the cells transfected
with the vector encoding wild-type USF-2a and cultured under normoxia,
PAI-1 mRNA expression was inhibited by about 70% (Figure
2A). Transfection of the USF-2a
expression vector reduced the hypoxia-dependent PAI-1 mRNA induction by
about 60%. However, the mRNA levels were still about 2-fold higher
than in the USF-2a-transfected cells cultured under normoxia. This
inhibition of PAI-1 mRNA expression was not observed in cells
transfected with the HU2a vector.
The USF-mediated decrease of PAI-1 mRNA was followed by a decrease of
PAI-1 protein. It was found that USF-2a-transfected hepatocytes
cultured under normoxia or hypoxia secreted about 50% less PAI-1
protein into the medium than the control cells (Figure 2A). However, as
observed with the PAI-1 mRNA, the amount of PAI-1 protein in the
USF-transfected cells measured under hypoxia was still enhanced by
about 2-fold compared with the PAI-1 levels under normoxia. Again, the
amount of PAI-1 protein in the medium of the cells transfected with the
To investigate that the inhibitory action of USF-2a is not due to the effect of the protein to form inactive homodimers, the PKL expression, which should be activated by USF,23 was examined. In the cells cultured under mild hypoxia and transfected with the empty control vector, PKL mRNA levels were about 3.5 times higher than under normoxia. The same was found for the PKL protein (Figure 2A, lower panel). In the cells transfected with the USF-2a expression vector, the PKL mRNA levels were increased by USF-2a by about 2.5 fold under normoxia and by about 4.5 fold under hypoxia, and the PKL protein level was enhanced by about 3.5 fold under normoxia and by about 5-fold under hypoxia (Figure 2). When the Inhibition of PAI-1 promoter-controlled Luc expression and induction of PKL promoter-controlled Luc expression by overexpression of USF-2a under both normoxia and mild hypoxia To further substantiate that the binding of USF-2a to the HREs, as revealed by EMSA (Figure 1), may be involved in the regulation of PAI-1 expression not only under normoxic but also under hypoxic conditions, primary hepatocytes were cotransfected with the hypoxia responsive Luc gene construct pGl3PAI-76614 and either control pCMV plasmid, USF-2a, orHU2a vectors. In hepatocytes transfected with
pGl3PAI-766, Luc activity was about 2.5-fold higher at 8% O2 than at 16% O2, in line with a previous
study.14 In hepatocytes cotransfected with pGI3PAI-766 and
USF-2a, the Luc activity was decreased by about 8-fold under both
normoxia and hypoxia (Figure 3). Thus,
USF-2a cotransfections did not abolish induction of Luc activity under
hypoxia. The Luc activity in pGl3PAI-766- and HU2a-cotransfected
hepatocytes did not differ significantly from the control
values.
Again, to further corroborate the positive effect of USF-2a on
PKL expression, hepatocytes were cotransfected with the USF expression vectors and a PKL promoter Luc construct
(pGl3PKL-183) that was shown to be activated by
USF.23 The cells were then cultured under normoxia or mild
hypoxia. In hepatocytes transfected with pGl3PKL-183 and
the pCMV control vector, hypoxia elicited only a marginal induction of
Luc activity. In the USF-2a- and pGl3PKL-183-cotransfected cells, Luc activity was
increased by about 3-fold under both normoxia and hypoxia. By contrast,
Thus, PAI-1 mRNA protein as well as PAI-1-promoter controlled Luc expression in primary rat hepatocytes cultured under normoxia and hypoxia were inhibited by overexpression of USF-2a. Inhibition of transfected rat PAI-1 promoter Luc gene constructs by wild-type and mutant USF-2a To determine what domains of USF-2a were involved in the regulation of PAI-1 gene expression, the wild-type rat PAI-1 promoter Luc construct pGl3PAI-766 and plasmids expressing wild-type USF-2a, the mutant proteinHU2a, and the mutant TDU2, which lacks the first
198 amino acids of the transactivation domain, were cotransfected. In
hepatocytes cotransfected with pGI3PAI-766 and USF-2a, the Luc activity
was decreased (Figure 3). The Luc activity in pGl3PAI-766- and
HU2a-cotransfected hepatocytes was about the same as in the control.
In hepatocytes cotransfected with pGI3PAI-766 and the TDU2 vector,
which contains the intact DNA-binding domain,23 the Luc
activity was decreased by about 5-fold (Figure 3). In contrast, after
cotransfection of the TDU2 vector and the pGl3-183PKL construct, the Luc activity was in the range of the controls (Figure 3). These data demonstrated that only the bHLH-zip domain but not the
N-terminal transactivation domain was necessary for the USF-2a-dependent inhibition of PAI-1 expression in primary
hepatocytes, whereas PKL promoter activation by USF-2
required the transactivation domain.
Cotransfection of the plasmid pGl3PAI-276 containing a 276-bp fragment
of the rat PAI-1 promoter in front of the Luc gene with the USF-2a
plasmid resulted in the same inhibition of Luc activity as with the
pGl3PAI-766 Luc construct. (Figure 4).
The inhibition was not observed when the pGl3PAI-276 construct was cotransfected with the
Mutations of the HRE sequences in PAI-1 Luc gene constructs abolished the inhibition of Luc activity To investigate the role of the HRE-1 and HRE-2 sequences in the USF-2a-dependent inhibition of PAI-1 expression, the wild-type PAI-276 promoter Luc construct and PAI-276 promoter Luc constructs with either a mutated HRE-1 or a HRE-2 were cotransfected with the expression vectors for USF-2a,HU2a, or the control pCMV into primary
hepatocytes (Figure 4). Mutation of the nucleotides 175/170
encompassing the HRE-1 site in the construct pGl3PAI-276M1 resulted in
a strong (about 12-fold) induction of Luc activity compared with
pGl3PAI-276-transfected hepatocytes. In cotransfection experiments,
USF-2a acted as inhibitor and reduced the Luc activity by about 7-fold,
but the overall Luc activity was about 5-fold higher than the wild-type
pGl3PAI-276 control. Cotransfection of pGl3PAI-276M1 and HU2a led to
the maximal observed (about 15-fold) induction of Luc activity
(Figure 4).
When the nucleotides Competition by HIF-1 expression vectors together with the PAI-1 promoter Luc
constructs pGl3PAI-766, pGl3PAI-766M1, and pGl3PAI-766M2 were performed.
Cotransfection of pGl3PAI-766 with the USF-2a vector again reduced Luc
activity by about 8-fold, whereas cotransfection of the HIF-1
In hepatocytes transfected with the HRE-2-mutated construct
pGl3PAI-766M2 and the USF-2a vector, Luc activity was diminished by
about 4-fold. As shown,14 cotransfection of pGl3PAI-766M2 and the HIF-1
In this study it was demonstrated that the transcription factor USF-2a acted as inhibitor of the rat PAI-1 gene expression via binding to the noncanonical E-box sequence HRE-1. The inhibitory effect of USF-2a on the PAI-1 gene expression was due to the DNA-binding domain of USF-2a and not exerted by the transactivation domain. The HRE-1 is separated by only 4 bp from the other E-box-like sequence in the rat PAI-1 promoter, HRE-2, which could bind HIF-1 acting as the activator of the rat PAI-1 expression under mild hypoxia. Overexpression of USF-2a in general inhibited the expression of PAI-1 but did not interfere with the induction of PAI-1 expression by mild hypoxia per se. The upstream stimulatory factors as inhibitors and activators The finding that USF inhibited PAI-1 production in hepatocytes was at a first glance unexpected because USF was originally identified from HeLa cell nuclei as an activator of the adenovirus major late promoter.16 However, as in this study, USF was able to act as an inhibitor of gene expression. Using chromatin cross-linking and immunoprecipitation protocols, it was demonstrated that both c-Myc and USF bound to exactly the same E-box site on the cad (carbamoyl-phosphate synthase/aspartate carbamoyltransferase/dihydroorotase) promoter in cultured B5-4 cells.33,34 Binding of USF and competition with TFE3 at the microE3 box within the immunoglobulin heavy chain enhancer lead to inhibition of enhancer activity in NIH3T3 cells.35 Furthermore, USF was also shown to compete with the arylhdrocarbon receptor/arylhdrocarbon receptor nuclear translocator (AHR-ARNT) heterodimer for binding to the xenobiotic-responsive element (XRE) in the rabbit CYP1A1 gene,36 thereby attenuating the AHR-ARNT mediated activation of XRE-TK/Luc gene constructs in RK13 cells. Moreover, USF repressed XMyoD binding to the XMyoDa promoter, thereby preventing XMyoDa autoactivation in 10T[1/2] cells.37 Thus, the findings of this study with the PAI-1 gene are another example for the role of USF as an inhibitor of gene expression.Despite the fact that in vivo most of the USF proteins exist as
heterodimers,19 one might expect that the USF-2 homodimers formed after overexpression of USF-2 alone could result in a squelching mechanism, thereby mediating the suppression via an indirect effect. However, homodimers formed after overexpression of USF-1 or USF-2 in
HeLa or hepatoma cells were shown to activate a promoter containing 4 copies of an E-box or the The PAI-1 HREs as USF binding sites: competition between USF and HIF In a previous study it was shown that the PAI-1 promoter region177/152 contains 2 E-box sequences 5'-CACGTA-3' (175/170) and
5'-CACGTG-3' (165/160), which were named HRE-1 and HRE-2, respectively. The induction of PAI-1 gene expression by hypoxia was
shown to depend on both the HRE-1 and HRE-2. Gel shift analysis indicated that HRE-2 bound the HIF-1. In contrast, the oligonucleotide spanning the HRE-1 was able to bind a complex, formation of which was
independent from the pO2.14 Thus,
it was proposed that other partners of the bHLH-protein family such as
USF or Myc may interact at the HRE-1 of the PAI-1 promoter. Indeed,
this study showed binding of USF-1 and USF-2 to both HREs of the PAI-1
promoter. In line with the present findings, it was shown during this
study that with nuclear extracts obtained from serum-stimulated renal epithelial cells (NRK-52E, clone EC-1) the HRE-2 can be bound by USF-1,
whereas the HRE-1 sequence was not investigated; but, again, incubation
of NRK-52E nuclear extracts with antibodies to Myc or Max
failed to produce a supershift with the HRE-2
oligonucleotide.43
The gel shift and supershift assays demonstrated that USF-2 bound with
high affinity to the HRE-1 and to a lesser extent to the HRE-2. Because
the preferred E-box core sequence for USF is 5'-CACGTG-3',16,44 one would have expected that the
HRE-2 functions as the predominant USF binding site within the PAI-1
promoter. Indeed, supershift assays and the cotransfection experiments
of pGl3PAI-276M1 in which the HRE-2, but not the HRE-1, was intact with
an expression vector for USF-2a caused inhibition of Luc activity
(Figures 1, 4). However, this inhibition was not as strong as with the
wild-type pGl3PAI-276 construct or with the HRE-2-defective plasmid
pGl3PAIM2, indicating that HRE-1 appeared to be the primary USF binding
site. Thus, the finding that USF bound the imperfect E-box HRE-1
5'-CACGTA-3' in the PAI-1 promoter is also in good agreement with
previous reports in which USF has been shown to bind the imperfect
E-boxes 5'-CCCGTG-3' as in the rat In the cotransfection experiments with the vectors encoding a USF-2a
protein lacking DNA-binding activity ( The DNA-binding domain-dependent inhibitory action of USF-2 on the
PAI-1 gene expression might play a role in the regulation of PAI-1
expression in response to diverse stimuli. Because the HRE-2 bound USF
as well as HIF-1, it makes this site attractive to a competitive
regulation by the DNA binding of both transcription factors. This was
indeed shown with the cotransfection experiments with the USF-2a and
HIF-1 Role of the PAI-1 inhibition by USF in growth progression and carcinogenesis The finding of this study that USF inhibited PAI-1 gene expression may have an important role during growth and regeneration processes. It was suggested that when cells progress from quiescence into the S phase, the transcription factor Myc/Max heterodimer competed with USF proteins for binding to the E-box.33 Accordingly, USF-1 and USF-2 mediated antiproliferative properties; when overexpressed in REF cells they inhibited c-Myc-induced cellular transformation.52 The Myc-dependent cellular transformation was inhibited only when wild-type USF-1 and USF-2 or vectors for USF mutants lacking the N-terminal transactivation domain were transfected. The DNA-binding-deficient forms of USF-1 and USF-2 did not affect the Myc-dependent foci formation in REF cells.52 This is in line with the results of the present study in which the cotransfection experiments with the USF-2a mutants lacking the DNA-binding domain (HU2a) and the PAI-1 Luc constructs
demonstrated the necessity of the DNA-binding domain for the inhibitory
action of USF (Figure 3). On one hand, the results support that the
role of USF-2 does not appear to be cell-specific because USF-2 also
exerted a strong inhibition of E1A-mediated transformation of REF cells
and a strong suppression of HeLa cell colony formation52;
however, on the other hand, in the Saos-2 osteosarcoma cell line, USF
proteins had no effect on cell proliferation, suggesting a model
involving a cell type-specialized coactivator.53
The antiproliferative activities of USF suggested that inactive USF could promote carcinogenesis. Indeed, in several cell lines from breast tumors USF was completely inactive.54 Thus, the loss of the repression by USF with the PAI-1 promoter gene constructs with the mutated USF site and the subsequent higher PAI-1 Luc expression as shown in Figure 4 would also coincide with the observation that PAI-1 was found to be expressed at higher levels in breast and other cancer cells55; it may be necessary for optimal metastasis formation as shown for cultured lung cancer cells.56 Moreover, in a number of clinical studies it was demonstrated that high PAI-1 levels in patients suffering from various types of cancer indicated a poor prognosis.57-59 Thus, it appears that USF controls the production of metabolic enzymes such as fatty acid synthetase,41 PKL60 and, as shown in this study, inhibitors of proteolysis such as PAI-1, which contributes to the control of cell proliferation and tumor growth.
We thank Dr A. Kahn and Dr M. Raymondjean (Institut Cochin de
Genetique Moleculaire, Universite Rene Descartes, Paris) for the
kind gift of the
Submitted July 6, 2000; accepted January 2, 2001.
Supported by the Deutsche Forschungsgemeinschaft SFB 402 Teilprojekt A1 and GRU 335.
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: T. Kietzmann, Institut für Biochemie und Molekulare Zellbiologie, Humboldtallee 23, D-37073 Göttingen, Germany; e-mail: tkietzm{at}gwdg.de.
1. Lijnen HR, Collen D. Mechanisms of plasminogen activation by mammalian plasminogen activators. Enzyme. 1988;40:90-96[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.
1986;261:11207-11213 3. 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. 1997;150:579-590[Abstract]. |
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Abstract
Introduction
