1 1. Medizinische Klinik, Klinikum rechts der Isar und Deutsches Herzzentrum,
Lazarettstraße 36, 80636 München, Germany
2 Institut für Klinische Chemie und Pathobiochemie, Technische
Universität München, Ismaningerstraße 22, 81675 München,
Germany
3 Institut Albert Bonniot, Joseph Fourier University of Grenoble, Faculty of
Medicine, Domaine de la Merci, 38706 La Tronche Cedex, France
4 Laboratorium für Molekulare Biologie, Genzentrum der Universität
München, Feodor Lynen Strasse 25, 81377 München, Germany
5 Department of Obstetrics, Gynecology and Reproductive Sciences, University of
California San Francisco Comprehensive Cancer Center, San Francisco, CA
94143-0875, USA
* Author for correspondence (e-mail: gawaz{at}dhm.mhn.de)
Accepted 1 August 2002
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Summary |
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Key words: ß3-endonexin, ß3-integrins, NF-B, uPAR, Angiogenesis
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Introduction |
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Recently, important links have been identified between
ß3-integrins and proteases, which play a pivotal role in the
regulation of cell migration. One important protease involved in these
processes is the serine protease urokinase-type plasminogen activator (uPA,
urokinase), which binds to the specific receptor uPAR. Urokinase converts
plasminogen to plasmin, a serine protease with broad substrate specificity for
several components of the ECM, including vitronectin, laminin and fibronectin
(Blasi, 1999;
Liotta et al., 1981
;
Schwartz et al., 1995
).
Binding of urokinase to uPAR increases the rate of plasmin formation at the
plasma membrane and focuses the proteolytic activity onto the leading edge of
tumor cells (Blasi, 1999
;
Ellis, 1996
).
Expression of uPAR is controlled mainly at the transcriptional level
although post-transcriptional regulation and recycling of uPAR to the plasma
membrane represent additional levels of regulation
(Conese and Blasi, 1995;
Lengyel et al., 1996
;
Lund et al., 1995
;
Shetty et al., 1997
;
Soravia et al., 1995
). The
importance of transcriptional regulation of the uPAR promoter by activator
protein 1 (AP-1), AP-2, Sp1 and NF-
B transcription factors and their
corresponding binding sites in the 5'-flanking site of the uPAR gene
have been recently defined (Allgayer et
al., 1999
; Lengyel et al.,
1996
; Soravia et al.,
1995
; Wang et al.,
2000
). Recent studies have indicated that the expression of uPAR
is linked to the expression and ligation of
vß3
(Hapke et al., 2001a
). Besides
the physical interaction of ß3-integrin and uPAR at the cell
surface, ß3-mediated outside-in signalling has been implicated
in the regulation of uPAR gene transcription, suggesting a mutual regulation
of adhesion and proteolysis (Chapman and
Wei, 2001
; Hapke et al.,
2001a
; Xue et al.,
1997
). Recently,
vß3-mediated
adhesion of human ovarian cancer cells on vitronectin has been shown to
downregulate uPAR that was associated with an increase in NF-kB activity
(Hapke et al., 2001b
). Cells
adhering to vitronectin, but not to fibronectin, have been found to
co-localize the urokinase-type plasminogen activator receptor (uPAR) to focal
adhesion contacts (Xue et al.,
1997
). Despite these indications that ß3-integrins
are regulating the expression of the uPAR through a PEA3/ets site in the uPAR
promoter (Hapke et al.,
2001a
), the molecular mechanism is poorly understood. While we
identified the regulation of uPAR by ß3-integrins and the
involvement of ß3-endonexin, the mechanism mediating the
signal downstream from the integrin receptor to the nucleus was still unclear.
To delineate this mechanism further, we have tested the role of the
cytoplasmic integrin binding protein ß3-endonexin on uPAR
expression. We report that ß3-endonexin binds to the
transcription factor NF-
B, inhibits interaction with its corresponding
promoter binding site, and subsequently inhibits uPAR transcription.
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Materials and Methods |
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Vectors, transfection and reporter analysis
Both isoforms of ß3-endonexin were cloned from a natural
killer cell cDNA library through amplification by PCR as described
(Gawaz et al., 2001).
ß3-endonexin mutants with deletion of the putative
K62RKK nuclear import sequence were generated in a two-step PCR
strategy (Gawaz et al., 2001
).
All constructs were identified by restriction digestion, purified by CsCl
centrifugation and verified by DNA sequencing before transfection. The uPAR
chloramphenicol acetyltransferase (uPAR-CAT-398) reporter, stretching from
-398 to +51 bp relative to translation start
(Hapke et al., 2001a
;
Lengyel et al., 1996
) was a
kind gift from Ernst Lengyel (University of California, San Francisco). The
NF-
B-mutated CAT-construct (uPAR-CAT mt-45) that has a mutation within
the NF-
B motif of the uPAR promoter
(Lengyel et al., 1996
;
Wang et al., 2000
) was kindly
provided by D. Boyd (Anderson Cancer Center, Houston, TX). The cDNA constructs
were expressed in endothelial cells or CHO cells by liposome-mediated
transfection (Superfect, Qiagen). Twenty-four hours before transfection, cells
were plated on 6-well culture plates. A total of 4 µg cDNA and 5 µl of
Superfect (Qiagen) reagent were incubated at room temperature for 10 minutes
in 75 µl of unsupplemented DMEM or M-199, respectively. 600 µl of
supplemented medium was then added and the DNA-Superfect complexes overlaid
onto the cells. The cells were incubated for 2 hours at 37°C, washed with
phosphate-buffered saline, and then incubated at 37°C with complete
medium. Medium was changed after 24 hours and the cells were analyzed at 48
hours. For the CAT-assays all transient transfections were performed in the
presence of 1 µg of uPAR-CAT reporter construct, 1 µg of a luciferase
expression vector and, if not otherwise indicated, 3 µg of expression
plasmids encoding the respective GFP-ß3-endonexin-isoform or
equimolar amounts of the control vector. CAT-assays were performed as
previously described (Lengyel et al.,
1996
). The cells were harvested and then lysed by repeated
freeze-thaw cycles in 0.25 M Tris-HCl (pH 7.8). Transfection efficiencies were
determined by the luciferase activity assay. After normalization for
transfection efficiency, CAT activity was measured by incubation of cell
lysates at 37°C with 4 µM [14C]chloramphenicol and 1 mg of
acetyl coenzymA/ml. The mixture was separated by extraction with ethyl acetate
and acetylated products were separated on thin layer chromatography plates
using chloroformmethanol as the mobile phase. The radioactive dots were
visualized by autoradiography and were quantified using a BioRad GelDoc
scanning software.
Cell lysis and Immunoblot analysis
Cells were lysed in a buffer containing 20 mM Tris (pH 7.2), 300 mM NaCl, 2
mM EGTA, 2% Triton X-100, 2% sodiumdesoxychalat, 0.2% SDS, 1 mM aprotinin, 0.1
mM leupeptin and 5 mM PMSF. After incubating for 30 minutes on ice, the
samples were centrifuged at 13,000 g to remove insoluble
material. To distinguish between cytosolic and nuclear fractions cells were
incubated in a cytosolic buffer containing 10 mM Hepes (pH 7.9), 10 mM KCl,
300 mM sucrose, 1.5 mM MgCl2, 0.5 mM DTT, 0.5 mM PMSF and protease
inhibitors for 5 minutes. Thereafter the nuclear pellet was washed once and
then resuspended in a buffer containing 20 mM Hepes (pH 7.9), 0.1 M KCl, 0.1 M
NaCl, 0.5 mM DTT, 0.5 mM PMSF and 0.2 M glycerol. Pellets were frozen and
thawed three times, cleared by centrifugation and the nuclear fraction was
aliquoted and stored at -70°C. Total protein content was determined by
using standard colorimetric assays (Bio-Rad, Munich); normalized aliquots of
those samples have subsequently been employed. The His-tagged recombinant
endonexin-short (14 kDa) was produced and purified according to standard
protocols. Proteins were separated on SDS-PAGE and transferred to Immobilion-P
membranes (Millipore, Bedford, MA). The filter was blocked for 1 hour in 5%
nonfat dry milk in PBS-Tween. Immunodetections were performed using sequential
antibodies as described followed by horseradish peroxidase-conjugated
secondary antibodies (Vector Laboratories, Burlingame, CA) and
chemiluminescence (Amersham Pharmacia Biotech).
Immunoprecipitation
For immunoprecipitation (IP), transiently transfected endothelial cells
were lysed as described above. Equilibrated agarose A (Roche Diagnostics,
Mannheim, Germany) was incubated with 2 µg/ml p65 mAb or GFP pAb for 1 hour
at room temperature (RT). 200 µg cell lysate was added in IP-buffer [20 mM
Tris pH 7.4, 200 mM NaCl, 1 mM DTT, 0.5 mM AEBSF
(4-(2-aminoethyl)-benzolsulfonylfluorid) and protease inhibitors] for 4 hours
at 4°C. Immunoprecipitates were washed five times in IP-buffer prior to
dissociation in SDS sample buffer.
Pull-down assay
The En-S and En-L cDNA was inserted into the SalI and
NotI cloning sites in pGEX4T3 (Amersham Pharmacia Biotech).
Glutathion-S-transferase (GST) or GST fusion proteins were expressed in E.
coli BL21 cells, purified and immobilized on Glutathion Sepharose beads
(Amersham Pharmacia Biotech) for 20 minutes at RT. To precipitate recombinant
human p50 (rhNF-B, Promega, Mannheim, Germany) the coated beads were
incubated with 3 µg of the protein in a binding buffer (20 mM Hepes pH 7.9,
200 mM NaCl, 0.25 mM MgCl2, 0.5 mM DTT, 1% NP-40, 1 mM ATP, 1 mM
GTP and 0.1 mM CaCl2) for 3 hours at 4°C. For
B
oligonucleotide competition studies (
B consensus, 5'-GAT CTG GGA
ACT TCC AAA GC-3';
B consensus mutant, 5'-GAT CTG GTC CCT
TCC AAA GC-3'), the protein was preincubated with the oligonucleotides
at a concentration of 3.5 µM for 30 minutes at RT before adding the
GST-beads. With the
B uPAR oligonucleotide, which contains the
NF-
B sequence (5'-ACG TCT GGG AGG AGT CCC TGG-3') of the
uPAR promoter (Wang et al.,
2000
), a concentration of 3.5 and 10.5 µM oligonucleotide has
been used. After five washes with binding buffer the bound proteins were
dissociated in SDS sample buffer and analyzed by immunoblotting.
Electrophoretic mobility shift assay (EMSA)
Nuclear extracts from interleukin-1ß (IL-1ß, 100
pg/ml)-stimulated HUVECs were prepared as described above. 2-4 µg were
incubated with raising ratios (1:2, 1:3, 1:4) of the GST-fusion proteins in a
binding buffer containing 40 mM Hepes (pH 7.9), 100 mM KCl, 2 mM DTT, 1 mM
EDTA, 10% glycerol, 2 mg/ml BSA, 0.2% NP-40, and 50 ng/µl poly(dI/dC) for
60 minutes at 4°C. 105 cpm of a Klenow end-labeled
([-32P]dCTP) prototypic double-stranded Ig
-chain
oligonucleotide (Brand et al.,
1996
) was added to each reaction mixture and binding was allowed
at RT for 30 minutes. Samples were run in 0.25xTBE buffer with loading
dye on non-denaturing 5% polyacrylamid gels at 125 V for 3 hours. The gel was
dried and exposed to X-ray film overnight at -80°C. In another EMSA the
nuclear extract was replaced by gel shift units of recombinant p50
(rhNF-
Bp50, Promega) according to the manufacturer's instructions.
Flow cytometry and confocal laser immunofluorescence microscopy
Transient transfectants were harvested in citric saline containing 0.13 M
KCl and 150 mM sodium citrate. After fixation by adding an equal volume of 1%
paraformaldehyde in PBS, cells were collected by centrifugation at 550
g for 5 minutes and washed once in PBS. For uPAR staining,
fixed cells were resuspended in 100 µl PBS containing 5 µg/ml anti-uPAR
mouse-IgG. After 30 minutes of incubation cells were washed twice and
resuspended again in 100 µl PBS containing phytoerythrin (PE)-conjugated
anti-mouse IgG1 mAb (50 µg/ml). After a further 30 minutes
incubation in the dark, cells were washed twice and resuspended in 0.5 ml
PBS/0.5% paraformaldehyde and analyzed by flow cytometry on a FACScan (Becton
Dickinson). 10,000 cells were analyzed for red (PE) immunofluorescence. For
confocal laser immunofluorescence microscopy, transfected cells were
cultivated on coverslips coated with vitronectin (5 µg/ml) at 4°C
overnight and blocked for 1 hour with 5% BSA in PBS at room temperature. Cell
monolayers were then fixed and covered with mounting medium.
Immunofluorescence analysis was performed using a Leica confocal laser
microscope equipped with a TCS software program.
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Results |
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To evaluate the effect of ß3-endonexin on uPAR expression,
endothelial cells were transiently transfected with GFP-fusion proteins
GFP/En-S or GFP/En-L and uPAR protein expression was determined by flow
cytometry. Transfection with ß3-endonexin resulted in a
substantial decrease in uPAR protein expression when compared with
mock-transfected control samples. In En-S-transfected cells, uPAR protein
expression was reduced by 45%, whereas in En-L-transfected cells uPAR
expression was reduced by 28% (Fig.
1B). Next, we evaluated whether this reduced uPAR protein
expression is paralleled by a reduction in uPAR transcription. As the 398 bp
fragment in the 5'-flanking region of the uPAR promoter is sufficient
for the transcriptional activity of the uPAR gene, we used a -398 bp uPAR
promoter-driven CAT construct for transcriptional assays. Cells were
cotransfected with the uPAR-CAT reporter along with various amounts of an
expression vector that encoded En-L and En-S
(Fig. 2A,B). As described for
the uPAR protein, CAT activity driven by the uPAR promoter was substantially
inhibited by the short form (50%), whereas the long form of
ß3-endonexin resulted in less reduction in uPAR promoter
activity (
30%) (Fig.
2A,B). Taken together, the data shown in
Fig. 1B and
Fig. 2 demonstrate that the
integrin-binding cytoplasmic protein ß3-endonexin
downregulates uPAR protein expression and promoter activity.
|
ß3-endonexin localizes to the cell nucleus through a
process mediated by the nuclear localization sequence K62RKK
To evaluate the mechanism of ß3-endonexin on uPAR gene
transcription we investigated the cellular location of
ß3-endonexin. Both forms of ß3-endonexin were
expressed by transient transfection in endothelial cells as GFP-fusion protein
and the subcellular distribution was visualized by confocal laser scanning
microscopy. As shown in Fig.
3A, the control GFP protein was distributed throughout the cell,
whereas GPF/En-S and GFP/En-L localized primarily to the nucleus. In addition,
GFP/En-S was found in the cytoplasm and the plasma membrane (data not shown).
Identical results were obtained by analysing cellular subfractions by
immunoblot analyses for both the GFP-fusion protein
(Fig. 3B) and the endogenous
form (M.G. and F.B., unpublished). The nuclear appearance of
ß3-endonexin is in accordance with a putative nuclear
localization signal sequence K62RKK
(Shattil et al., 1995). To
further characterize the importance of this nuclear import sequence we
constructed a mutant form of ß3-endonexin that has a deletion
in the K62RKK nuclear import sequence
(Gawaz et al., 2001
). As shown
in Fig. 3A and B, deletion of
the K62RKK signal sequence almost completely abolished the nuclear
location of ß3-endonexin, indicating that K62RKK is
required for nuclear import of ß3-endonexin. These results
suggest that ß3-endonexin is both a cytoplasmic and a nuclear
protein.
|
The NF-B-binding site of the uPAR promoter is required for
ß3-endonexin-mediated downregulation of uPAR gene
transcription
ß3-integrins mediate through their cytoplasmic domain
signals into the cell and affect gene regulation such as the induction of cell
cycle progression via Ras-mitogen-activated protein kinase (MAPK) pathway
(Chen et al., 1994;
LaFlamme et al., 1997
). By
testing dominant-negative expression vectors for several integrin-mediated
pathways we could not find any involvement of these integrin-mediated events
in uPAR gene regulation (E.L., unpublished). The downregulation of the uPAR
promoter by ß3-integrin is mediated in part through a
PEA3/ets motif, and other sequences in the promoter region -202 bp
relative to the translation start play a role in this regulation. We therefore
investigated several binding sites in this area
(Hapke et al., 2001a
).
Recently, a NF-
B binding site at -45 bp was described in the uPAR
promoter, which is required for uPAR promoter activity
(Lengyel et al., 1996
;
Soravia et al., 1995
;
Wang et al., 1995
;
Wang et al., 2000
). Because
the constitutive activity of the uPAR promoter is driven at least in part
through the NF-
B motif, we were interested in the role of the
NF-
B sequence in the ß3-endonexin-dependent regulation
of the uPAR promoter (Lengyel et al.,
1996
; Wang et al.,
2000
). We co-transfected cells with both isoforms of
ß3-endonexin and a CAT-reporter construct driven by the -398
bp uPAR promoter sequence mutated at the NF-
B motif (uPAR-CAT mt-45)
(Wang et al., 2000
). In
accordance with Wang et al., the activity of the NF-
B-mutated
promoter-construct was decreased in comparison with the unmutated promoter
(Wang et al., 2000
)
(Fig. 4A). In contrast to the
wild-type uPAR promoter (Fig.
2A,B; Fig. 4A),
co-transfection of ß3-endonexin with the mutated uPAR promoter
did not show a further significant inhibition of the uPAR promoter activity
(Fig. 4A), implying that the
NF-
B motif is critical in the ß3-endonexin-dependent
downregulation of uPAR transcription.
|
ß3-endonexin inhibits NF-B binding
activity
In view of the previous results we considered that
ß3-endonexin might have DNA-binding activity and directly
interfere with binding to the B motif. We constructed and purified a
recombinant GST/ß3-endonexin fusion protein and evaluated
DNA-binding activity with EMSA under several experimental conditions using
oligonucleotides containing a
B consensus sequence
(Brand et al., 1996
). We did
not find any specific DNA-binding activity neither of the short nor the long
ß3-endonexin isoform (F.B., unpublished). Therefore, we
speculated that ß3-endonexin might modulate
NF-
B-dependent uPAR expression by direct interference with the p50/p65
complex. To test this hypothesis, we performed
B-DNA gel-shift assays
with nuclear extracts isolated from IL-1ß-activated endothelial cells.
Recombinant GST/ß3-endonexin was added in increasing amounts
to nuclear extracts and NF-
B-binding activity was determined by EMSA
(Fig. 4B). In the presence of
En-S, p50/p65-binding activity was inhibited in a dose-dependent manner. In
comparison, we found less reduction when En-L was added to the nuclear
extracts or when the control GST protein was incubated with nuclear extracts
it did not substantially reduce NF-
B binding activity
(Fig. 4B). This result suggests
that ß3-endonexin inhibits NF-
B-DNA binding through
direct interference with the p50/p65 complex.
ß3-endonexin directly interacts with the p50/p65
complex
The experimental evidence above showed that downregulation of uPAR is
regulated by direct interaction of ß3-endonexin with the
NF-B complex. To further evaluate whether ß3-endonexin
physically interacts with the p50/p65 complex we performed
co-immunoprecipitation studies of endothelial cell extracts. Endothelial cells
were transiently transfected with GFP/En-S or GFP/En-L or the control GFP
vector and the GFP fusion proteins immunoprecipitated with a GFP mAb. The
immunoprecipitate was evaluated for the presence of the p50/p65 complex by
immunoblotting with anti-p65 mAb (Fig.
5A) and anti-p50 (F.B., unpublished). We found that both GFP/En-S
and GFP/En-L but not the GFP control precipitates p50 and p65 protein,
indicating that both isoforms of ß3-endonexin bind directly to
the endogenous p50/p65 complex. Identical results were obtained in the
opposite experiment, when p65 was first immunoprecipitated with an anti-p65
mAb and the precipitate was probed for the presence of
GFP/ß3-endonexin in immunoblots
(Fig. 5A).
|
To further confirm that ß3-endonexin directly interacts
with the NF-B complex, we performed a pull-down assay with purified p50
fusion protein. Recombinant GST/En-S, GST/En-L or the control GST-fusion
protein were immobilized on Sepharose beads and incubated with isolated p50
protein. The probes were assayed for p50 binding by immunoblotting. Both
GST/En-S and GST/En-L but not GST alone precipitated p50
(Fig. 5B). To evaluate the
functional significance of ß3-endonexin interaction with p50,
purified p50 was incubated with increasing concentrations of
GST/ß3-endonexin and DNA-binding activity of p50 was
determined by EMSA. As shown in Fig.
5C, DNA binding of p50 was significantly decreased in the presence
of both isoforms of ß3-endonexin indicating that
ß3-endonexin directly inhibits binding of p50 complexes to
B DNA. To evaluate whether interaction of ß3-endonexin
with p50 can be inhibited with double-stranded
B DNA, precipitation
studies were performed in the presence of
B and mutated
B
oligonucleotides that do not bind to the p50/p65 complex
(Gawaz et al., 1998
). In the
presence of consensus
B oligonucleotides, p50 could not be precipitated
by GST/ß3-endonexin, whereas equimolar amounts of the mutated
B DNA did not have this effect (Fig.
5D). These results provide strong evidence that the interaction of
ß3-endonexin with p50 inhibits the accessibility of the
NF-
B complex for
B DNA and thereby inhibits NF-
B-mediated
uPAR transcription.
Next, we tested whether the observed competition can also be detected with
the B uPAR promotor consensus sequence. Similarly to the
B
consensus sequence, we found that
B uPAR DNA prevented p50 binding to
ß3-endonexin in a concentration-dependent manner
(Fig. 5E).
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Discussion |
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Recently, mutational promoter analyses suggested that a region between -398
and -197 bp of the uPAR promoter is critical for
ß3-integrin-induced downregulation of uPAR promoter activity
and that a PEA3/ets motif at -246 bp is involved in
ß3-integrin and ß3-endonexin-short-induced
reduction (Hapke et al.,
2001a). Based on these previous results, we extended our studies
and detected that not just the short form but also the long form of
ß3-endonexin is inhibiting uPAR protein and transcriptional
activity in a dose-dependent manner. Moreover, we also found that a
NF-
B motif is involved in ß3-endonexin-induced
downregulation of uPAR. This NF-
B site at position -45 bp was just
recently defined (Wang et al.,
2000
). Mutation of the NF-
B motif decreased the binding of
transcription factor NF-
B and reduced the constitutive uPAR promoter
activity (Wang et al., 2000
).
We used this mutated promoter construct and found that, in contrast to the
wild-type promoter, ß3-endonexin did not inhibit uPAR
transcription under the control of the mutated promoter deficient at the
NF-
B site. This novel finding implies that the NF-
B binding
motif is required for ß3-endonexin-mediated downregulation of
uPAR expression. Kashiwagi et al.
(Kashiwagi et al., 1997
) and
the present study showed that both isoforms of ß3-endonexin
translocate into the cell nucleus and that deletion of the putative nuclear
import sequence K62RKK prevents nuclear translocation of
ß3-endonexin (this study). A role for
ß3-endonexin in nuclear function was demonstrated by Ohtoshi
et al., who showed that ß3-endonexin binds to cyclin A and
inhibits cyclin A-Cdk2 kinase activity
(Ohtoshi et al., 2000
).
Together, these results suggest a role for ß3-endonexin in
gene regulation.
To assess whether ß3-endonexin has direct DNA-binding
activity we generated a recombinant fusion protein
GST-ß3-endonexin and performed DNA-binding studies using
double stranded oligonucleotides with various B-DNA sequences and
gel-shift assays. Under these conditions we did not find any specific
DNA-binding activity (X. Author, unpublished), implying that
ß3-endonexin does not directly bind to the NF-
B site
within the uPAR promoter and thus does not directly regulate uPAR
transcription. In gel-shift experiments using nuclear extracts of activated
endothelial cells we found that recombinant GST-fusion proteins of both
isoforms of ß3-endonexin substantially inhibited
B-DNA
binding activity, which suggests that ß3-endonexin directly
binds to proteins of the NF-
B complex and thereby inhibits binding of
this complex to its promoter site. Consequently, we found in
immunoprecipitation studies that ß3-endonexin directly
interacts with NF-
B complexes because endogenous p65 could be
precipitated with GFP-ß3-endonexin and vice versa. These
results are further supported by pull-down assays that showed that purified
p50 interacts with recombinant ß3-endonexin. Finally, the
findings that ß3-endonexin almost completely inhibits
B-DNA binding to p50 and that
B but not a mutated form of
B oligonucleotides inhibits ß3-endonexin interaction
with p50 provide strong evidence that ß3-endonexin directly
interferes with the DNA-binding site of the NF-
B complex
(Ghosh et al., 1995
;
Muller et al., 1995
;
Urban and Baeuerle, 1991
) and
thereby inhibits sterically NF-
B-dependent uPAR promoter activity. This
mechanism might explain the recent findings that
vß3/vitronectin-induced downregulation of
uPA and uPAR in human ovarian cancer cells (OV-MZ-6) is paralleled by a
substantially reduced activity of NF-
B
(Hapke et al., 2001b
).
Our study provides a molecular link between
ß3-integrinmediated cell adhesion and uPAR expression and
suggests ß3-endonexin as a mediator between these two systems.
We identified crucial steps in the signal transduction pathway, between the
cell surface and the nucleus. Because ß3-endonexin is
localized in both the cytoplasm and nucleus it is tempting to speculate that
ß3-endonexin acts as a shuttle protein connecting stimulated
internalization of ß3-integrin with formation of a
ß3-endonexin/NF-B complex that results in
downregulation of uPAR expression. This may represent a pathway that is
independent of the established I
B kinase complex-mediated signalling
cascades (Israel, 2000
;
Karin and Ben-Neriah, 2000
),
although further investigations will be necessary for conclusive data.
Interestingly, we found (F.B., unpublished)
(Hapke et al., 2001a
) that
exclusively the ß3-A isoform but not the ß3-C
or the mutated ß3-A-Y759A form that is deficient in the
ß3-endonexin-binding motif NITY reduces uPAR expression in
transfected cells. Because ß3-A is internalized upon ligation
and ß3-endonexin regulates endocytosis of ß3-A
(Gawaz et al., 2001
) we have
now significant data that ß3-endonexin links
vß3-mediated endothelial adhesion with
downregulation of uPAR. Thus, the balance between available isoforms of
ß3-integrins and of ß3-endonexin might provide
a switch within the regulation of uPAR and the angiogenic phenotype of
endothelial cells. Further studies should reveal whether the
ß3-endonexin shuttle mechanism only affects constitutive gene
expression or is also involved in inducible gene regulation. It is also not
clear which stimuli may use this pathway and which additional target genes are
affected.
The present results elucidate novel mechanisms for how signals generated by
cell adhesion at the plasma membrane are communicated to the cell nucleus and
regulate gene expression. Examples of proteins that are involved in both cell
adhesion and transcriptional regulation are ß-catenin and JAB1
(jun-activation-domain-binding protein 1), which support the concept that
cytoplasmic shuttle molecules connect cell adhesion events with gene
regulation (Bianchi et al.,
2000; Willert and Nusse,
1998
). JAB1, a coactivator of the c-Jun transcription factor, was
found to be present both in the nucleus and in the cytoplasm of cells, and a
fraction of JAB1 colocalizes with the integrin
L/ß2
(LFA-1) at the cell membrane. LFA-1 engagement is followed by an increase in
the nuclear pool of JAB1, paralleled by enhanced binding of c-Jun-containing
AP-1 complexes to their DNA consensus site and increased transactivation of an
AP-1-dependent promoter (Bianchi et al.,
2000
).
ß3-endonexin is expressed ubiquitously while expression of
ß3-integrins is limited to certain cell types including
platelets, endothelial cells or smooth muscle cells
(Shattil et al., 1995). Thus,
it is obvious that ß3-endonexin has additional biological
functions other then mechanisms related to cell adhesion events. Only
recently, two proteins (theta-associated protein, TAP20; nuclear receptor
coactivator protein, NRIF3) have been cloned that have high homology with
ß3-endonexin (Li et al.,
1999
; Li et al.,
2001
; Tang et al.,
1999
). TAP20 enhances endothelial cell migration and in vitro tube
formation and modulates ß5-integrins
(Tang et al., 1999
). NRIF3 has
been reported to be 100% identical in the first 111 residues to En-S, and in
the first 161 residues to En-L (Li et al.,
1999
). NRIF3 mediates functional specificity of two nuclear
hormone receptors such as the thyroid hormone receptor and the retinoid X
receptor, whereas both isoforms of ß3-endonexin did not
(Li et al., 1999
). The facts
that ß3-endonexin has 100% homology to NRIF3 and that En-L
differs only in the C-terminal domain from NRIF3 but does not interact with
nuclear hormone receptors (Li et al.,
1999
) suggest that modifying the C-terminal domain of
ß3-endonexin alters the cellular function significantly. This
may also explain why En-L, which is 100% identical to En-S but 50 amino acids
longer, is not as effective in inhibiting
B-binding activity and
NF-
B-dependent downregulation of uPAR as En-S.
Taken together, our study provides strong evidence that
ß3-endonexin acts as an important cytoplasmic regulator of
NF-B-dependent expression of uPAR and
ß3-integrin-mediated adhesion. These results extend our
understanding of how ß3-integrin-mediated adhesion and
proteolytic activity of the uPAR system are regulated, a fundamentally
important concept for coordinating proteolysis, adhesion, migration and
angiogenesis at the right time and place.
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Allgayer, H., Wang, H., Gallick, G. E., Crabtree, A., Mazar, A.,
Jones, T., Kraker, A. J. and Boyd, D. D. (1999).
Transcriptional induction of the urokinase receptor gene by a constitutively
active Src. Requirement of an upstream motif (-152/-135) bound with Spl.
J. Biol. Chem. 274,18428
-18437.
Bianchi, E., Denti, S., Granata, A., Bossi, G., Geginat, J., Villa, A., Rogge, L. and Pardi, R. (2000). Integrin LFA-1 interacts with the transcriptional co-activator JAB1 to modulate AP-1 activity. Nature 404,617 -621.[CrossRef][Medline]
Blasi, F. (1999). Proteolysis, cell adhesion, chemotaxis, and invasiveness are regulated by the u-PA/u-PAR/PAI-1 system. Thromb. Haemost. 82,298 -304.[Medline]
Brand, K., Page, S., Rogler, G., Bartsch, A., Brandl, R.,
Knuechel, R., Page, M., Kaltschmidt, C., Baeuerle, P. A. and Neumeier, D.
(1996). Activated transcription factor nuclear factor-kappa B is
present in the atherosclerotic lesion. J. Clin. Invest
97,1715
-1722.
Chapman, H. A. and Wei, Y. (2001). Protease crosstalk with integrins: the urokinase receptor paradigm. Thromb. Haemost. 86,124 -129.[Medline]
Chen, Q., Kinch, M. S., Lin, T. H., Burridge, K. and Juliano, R.
L. (1994). Integrin-mediated cell adhesion activates
mitogen-activated protein kinases. J. Biol. Chem.
269,26602
-26605.
Clark, E. A. and Brugge, J. S. (1995). Integrins and signal transduction pathways: the road taken. Science 268,233 -239.[Medline]
Conese, M. and Blasi, F. (1995). The urokinase/urokinase-receptor system and cancer invasion. Baillieres Clin. Haematol. 8,365 -389.[Medline]
Dedhar, S. and Hannigan, G. E. (1996). Integrin cytoplasmic interactions and bidirectional transmembrane signalling. Curr. Opin. Cell Biol. 8, 657-669.[CrossRef][Medline]
Dejana, E. (1993). Endothelial cell adhesive receptors. J. Cardiovasc. Pharmacol. 21 Suppl. 1,S18 -S21.[Medline]
Eigenthaler, M., Hofferer, L., Shattil, S. J. and Ginsberg, M.
H. (1997). A conserved sequence motif in the integrin beta3
cytoplasmic domain is required for its specific interaction with
beta3-endonexin. J. Biol. Chem.
272,7693
-7698.
Ellis, V. (1996). Functional analysis of the
cellular receptor for urokinase in plasminogen activation. Receptor binding
has no influence on the zymogenic nature of pro-urokinase. J. Biol.
Chem. 271,14779
-14784.
Gawaz, M., Neumann, F. J., Dickfeld, T., Koch, W., Laugwitz, K.
L., Adelsberger, H., Langenbrink, K., Page, S., Neumeier, D., Schömig, A.
et al. (1998). Activated platelets induce monocyte
chemotactic protein-1 secretion and surface expression of intercellular
adhesion molecule-1 on endothelial cells. Circulation
98,1164
-1171.
Gawaz, M., Besta, F., Ylänne, J., Knorr, T., Dierks, H.,
Böhm, T. and Kolanus, W. (2001). The NITY motif of the
beta-chain cytoplasmic domain is involved in stimulated internalization of the
beta3 integrin A isoform. J. Cell Sci.
114,1101
-1113.
Ghosh, G., van Duyne, G., Ghosh, S. and Sigler, P. B. (1995). Structure of NF-kappa B p50 homodimer bound to a kappa B site. Nature 373,303 -310.[CrossRef][Medline]
Hapke, S., Gawaz, M., Dehne, K., Kohler, J., Marshall, J. F.,
Graeff, H., Schmitt, M., Reuning, U. and Lengyel, E. (2001a).
beta(3)A-integrin downregulates the urokinase-type plasminogen activator
receptor (u-PAR) through a PEA3/ets transcriptional silencing element in the
u-PAR promoter. Mol. Cell. Biol.
21,2118
-2132.
Hapke, S., Kessler, H., Arroyo de Prada, N., Benge, A., Schmitt,
M., Lengyel, E. and Reuning, U. (2001b). Integrin
alpha(v)beta(3)/vitronectin interaction affects expression of the urokinase
system in human ovarian cancer cells. J. Biol. Chem.
276,26340
-26348.
Hynes, R. O. (1992). Integrins: versatility, modulation, and signaling in cell adhesion. Cell 69, 11-25.[Medline]
Israel, A. (2000). The IKK complex: an integrator of all signals that activate NF-kappaB? Trends Cell Biol. 10,129 -133.[CrossRef][Medline]
Karin, M. and Ben-Neriah, Y. (2000). Phosphorylation meets ubiquitination: the control of NF-[kappa]B activity. Annu. Rev. Immunol. 18,621 -663.[CrossRef][Medline]
Kashiwagi, H., Schwartz, M. A., Eigenthaler, M., Davis, K. A.,
Ginsberg, M. H. and Shattil, S. J. (1997). Affinity
modulation of platelet integrin alphaIIbbeta3 by beta3-endonexin, a selective
binding partner of the beta3 integrin cytoplasmic tail. J. Cell
Biol. 137,1433
-1443.
LaFlamme, S. E., Homan, S. M., Bodeau, A. L. and Mastrangelo, A. M. (1997). Integrin cytoplasmic domains as connectors to the cell's signal transduction apparatus. Matrix Biol. 16,153 -163.[CrossRef][Medline]
Lengyel, E., Wang, H., Stepp, E., Juarez, J., Wang, Y., Doe, W.,
Pfarr, C. M. and Boyd, D. (1996). Requirement of an upstream
AP-1 motif for the constitutive and phorbol ester-inducible expression of the
urokinase-type plasminogen activator receptor gene. J. Biol.
Chem. 271,23176
-23184.
Li, D., Desai-Yajnik, V., Lo, E., Schapira, M., Abagyan, R. and
Samuels, H. H. (1999). NRIF3 is a novel coactivator mediating
functional specificity of nuclear hormone receptors. Mol. Cell
Biol. 19,7191
-7202.
Li, D., Wang, F. and Samuels, H. H. (2001).
Domain Structure of the NRIF3 family of coregulators suggests potential dual
roles in transcriptional regulation. Mol. Cell. Biol.
21,8371
-8384.
Liotta, L. A., Goldfarb, R. H., Brundage, R., Siegal, G. P., Terranova, V. and Garbisa, S. (1981). Effect of plasminogen activator (urokinase), plasmin, and thrombin on glycoprotein and collagenous components of basement membrane. Cancer Res. 41,4629 -4636.[Abstract]
Liu, S., Calderwood, D. A. and Ginsberg, M. H.
(2000). Integrin cytoplasmic domain-binding proteins.
J. Cell Sci. 113,3563
-3571.
Lund, L. R., Ellis, V., Ronne, E., Pyke, C. and Dano, K. (1995). Transcriptional and post-transcriptional regulation of the receptor for urokinase-type plasminogen activator by cytokines and tumour promoters in the human lung carcinoma cell line A549. Biochem. J. 310,345 -352.[Medline]
Muller, C. W., Rey, F. A., Sodeoka, M., Verdine, G. L. and Harrison, S. C. (1995). Structure of the NF-kappa B p50 homodimer bound to DNA. Nature 373,311 -317.[CrossRef][Medline]
Ohtoshi, A. and Otoshi, H. (2001). Analysis of beta3-endonexin mutants for their ability to interact with cyclin A. Mol. Genet. Genomics 266,664 -671.[CrossRef][Medline]
Ohtoshi, A., Maeda, T., Higashi, H., Ashizawa, S., Yamada, M. and Hatakeyama, M. (2000). beta3-endonexin as a novel inhibitor of cyclin A-associated kinase. Biochem. Biophys. Res. Commun. 267,947 -952.[CrossRef][Medline]
Schwartz, M. A., Schaller, M. D. and Ginsberg, M. H. (1995). Integrins: emerging paradigms of signal transduction. Annu. Rev. Cell Dev. Biol. 11,549 -599.[CrossRef][Medline]
Shattil, S. J., O'Toole, T., Eigenthaler, M., Thon, V., Williams, M., Babior, B. M. and Ginsberg, M. H. (1995). Beta 3-endonexin, a novel polypeptide that interacts specifically with the cytoplasmic tail of the integrin beta 3 subunit. J. Cell Biol. 131,807 -816.[Abstract]
Shetty, S., Kumar, A. and Idell, S. (1997). Posttranscriptional regulation of urokinase receptor mRNA: identification of a novel urokinase receptor mRNA binding protein in human mesothelioma cells. Mol. Cell. Biol. 17,1075 -1083.[Abstract]
Soravia, E., Grebe, A., de Luca, P., Helin, K., Suh, T. T.,
Degen, J. L. and Blasi, F. (1995). A conserved TATA-less
proximal promoter drives basal transcription from the urokinase-type
plasminogen activator receptor gene. Blood
86,624
-635.
Tang, S., Gao, Y. and Ware, J. A. (1999).
Enhancement of endothelial cell migration and in vitro tube formation by
TAP20, a novel beta 5 integrin-modulating, PKC theta-dependent protein.
J. Cell Biol. 147,1073
-1084.
Urban, M. B. and Baeuerle, P. A. (1991). The role of the p50 and p65 subunits of NF-kappa B in the recognition of cognate sequences. New Biol. 3,279 -288.[Medline]
Wang, Y., Dang, J., Johnson, L. K., Selhamer, J. J. and Doe, W. F. (1995). Structure of the human urokinase receptor gene and its similarity to CD59 and the Ly-6 family. Eur. J. Biochem. 227,116 -122.[Abstract]
Wang, Y., Dang, J., Wang, H., Allgayer, H., Murrell, G. A. and
Boyd, D. (2000). Identification of a novel nuclear
factor-kappaB sequence involved in expression of urokinase-type plasminogen
activator receptor. Eur. J. Biochem.
267,3248
-3254.
Willert, K. and Nusse, R. (1998). Beta-catenin: a key mediator of Wnt signaling. Curr. Opin. Genet. Dev. 8,95 -102.[CrossRef][Medline]
Xue, W., Mizukami, I., Todd, R. F., III and Petty, H. R. (1997). Urokinase-type plasminogen activator receptors associate with beta1 and beta3 integrins of fibrosarcoma cells: dependence on extracellular matrix components. Cancer Res. 57,1682 -1689.[Abstract]
Ylänne, J., Huuskonen, J., O'Toole, T. E., Ginsberg, M. H.,
Virtanen, I. and Gahmberg, C. G. (1995). Mutation of the
cytoplasmic domain of the integrin beta 3 subunit. Differential effects on
cell spreading, recruitment to adhesion plaques, endocytosis, and
phagocytosis. J. Biol. Chem.
270,9550
-9557.
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