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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 95 No. 4 (February 15), 2000:
pp. 1309-1316
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGYAU#0
Transcriptional activation of urokinase by the Krüppel-like
factor Zf9/COPEB activates latent TGF- 1 in vascular endothelial
cells
Soichi Kojima,
Shinichi Hayashi,
Kentaro Shimokado,
Yasuhiro Suzuki,
Jun Shimada,
Massimo P. Crippa, and
Scott L. Friedman
From the Laboratory of Molecular Cell Sciences, Tsukuba Life Science
Center, The Institute of Physical and Chemical Research (RIKEN),
Koyadai, Tsukuba, Ibaraki 305-0074, Japan; First Department
of Internal Medicine, Nihon University School of Medicine,
Ohyaguchi-kamimachi, Itabashi, Tokyo 173-0032, Japan; National
Cardiovascular Center Research Institute, Osaka 565-0873, Japan;
Laboratory of Molecular Genetics, DIBIT-H. S. Raffaele, Via Olgettina,
Milano 20132, Italy; Division of Liver Diseases, Mount Sinai Medical
Center, New York, New York.
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Abstract |
Understanding the regulation of genes controlling fibrinolysis and
matrix homeostasis is essential for elucidating the basis of tissue
repair. A recently described novel Krüppel-like factor, Zf9, is
up-regulated in acute liver injury in activated hepatic stellate cells.
Because Zf9 can be induced widely, its activity was examined in
vascular endothelium, a key cell in vascular injury. Zf9 is induced as
an immediate-early response gene in bovine aortic endothelial cells
(BAECs) following treatment with serum or phorbol ester. Zf9
transcriptionally activates urokinase plasminogen activator (uPA).
Recombinant Zf9-GST binds to wild-type but not mutated `GC-box'
motifs within the human uPA promoter ( 63 to 32), with greatest
affinity to the middle of 3 contiguous GC boxes. Transient transfection
of Zf9 drives transactivation of a full-length uPA promoter- and GC
box-construct, but not a uPA promoter-construct devoid of GC boxes.
Transactivation of uPA by Zf9 is also supported in Drosophila
S2 cells. Most importantly, transiently transfected Zf9 up-regulates
endogenous uPA messenger RNA and activity in BAECs, resulting in
increased bioactive transforming growth factor-beta (TGF- ) via
enhancement of proteolytic activation of the latent molecule.
Furthermore, concomitant expression of Zf9 and uPA proteins was
observed in arterial endothelial cells after balloon injury in rats,
suggesting a potential role of Zf9 in uPA expression not only in vitro
but also in vivo. These findings suggest a role of Zf9 in the injury
response by enhancing uPA synthesis and subsequent activation of latent
TGF-.
(Blood. 2000;95:1309-1316)
© 2000 by The American Society of Hematology.
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Introduction |
Coordinated gene expression is a key component of the
response to tissue injury. Classes of induced genes include
extracellular matrix proteins,1-4 growth factors/cytokines
such as transforming growth factor- (TGF-),1-6 and
proteases such as urokinase plasminogen activator (uPA).1-9
uPA was originally discovered as a fibrinolytic factor responsible for
conversion of plasminogen to plasmin in dissolving vascular
thrombosis.10,11 However, it also has a key role in tissue
remodeling,7,9,12 metastasis,13,14 apoptosis,14,15 and immunity14 either directly
or through activation of other enzymes including matrix
proteases9,13,16 and cytokines.5,17 uPA has
been implicated in the activation of latent TGF-. By activating
plasminogen to plasmin, uPA can stimulate proteolytic activation of
latent TGF- in several tissues and cell types especially in
pathogenic situations.15,17-25 TGF- in turn mediates key
components of the injury response. It down-regulates the inflammation,
modulates growth inhibition and differentiation, and enhances
extracellular matrix production in a variety of cell types.1-4 Vascular endothelial cells are a potent source of
uPA, and given their critical position in tissue remodeling, are a key
regulator of the injury response.6 Thus, control of
uPA/plasmin and thereby TGF- levels in endothelial cells is an
important event in the response to tissue injury.
Circulating levels of plasminogen are fairly constant, so cellular
plasmin levels are controlled primarily by plasminogen activator (PA)
activity. Cellular PA activity is, in turn, controlled not only by
regulation of uPA synthesis but also by levels of its receptor
(uPAR)7,11 and inhibitor (PAI-1).10 uPAR is essential for localizing the PA activity on the cell surface leading to
acceleration of plasminogen activation,7,11 whereas PAI-1 rapidly inhibits PA activity.10 Thus, the balance between
uPA, uPAR, and PAI-1 levels is critical for regulating the level of net
fibrinolytic activity.10,11 Regulation of uPA gene
expression is therefore a key control point of cellular PA activity. It
is known that uPA expression by vascular cells is up-regulated during the process of tissue repair in vivo.7,8,12 However, the molecular basis for injury-induced uPA gene expression is not completely understood. Several transcription factors have been reported
to participate in this response. These include LFB3/HNF,26 CRE-binding protein,7 Ets-1,27
PEA3,28-30 UEF1-4,28-30 AP-1,28-31 and NF- B32 demonstrated in the cultured cells and Egr-1
in injured rat aorta.33 Sp1, the initial Krüppel-like
zinc finger protein,34,35 is also shown to be another
important transcriptional activator of uPA in vitro through its
interaction with 3 contiguous GC boxes immediately upstream of the TATA
box in the uPA promoter.36,37
Zf9/COPEB/GBF (soon to be renamed KLF6) is a novel zinc finger
transcription factor recently cloned from liver, placenta, and
leukocyte complementary DNA (cDNA) libraries.38-41 The
molecule belongs to the family of Krüppel-like transcription
factors,42-52 all of which contain 3 contiguous
C2H2 zinc fingers at the carboxyl-terminal domain, and recognize either GC box motifs or CACCC motifs in responsive promoters.39-52 A proximal role for Zf9 in
response to tissue injury has been suggested by its rapid induction in activated hepatic stellate cells, the key fibrogenic cell
type in liver injury and repair.38,39 Moreover, Zf9
transactivates key genes comprising the injury response, including
collagen  1(I), TGF-1, and types I and II TGF- receptors in
hepatic stellate cells.39,53
Given its widespread expression and interaction with GC box
motifs,39-41 we have explored a potential role for Zf9 in
uPA gene expression by endothelial cells. We have identified
Zf9 messenger RNA (mRNA) and protein in cultured endothelial cells,
which are induced by serum or phorbol esters. Zf9 stimulates uPA
transcription and endogenous uPA activity in these cells, leading to
proteolytic activation of latent TGF-1. Furthermore, concomitant
expression of Zf9 and uPA proteins was induced in arterial endothelial
cells following carotid balloon injury in rats. The data suggest that Zf9 may have an important role in regulation of PA/plasmin activity and
TGF-1 activity in vascular endothelium.
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Materials and methods |
Materials
Phorbol myristate acetate (PMA) was purchased from Sigma (Chemical
Co, St Louis, MO). Rabbit polyclonal antibodies against rat Zf9 and rat uPA were obtained from Santa Cruz Biotechnology (Santa
Cruz, CA) and American Diagnostica (Greenwich, CT),
respectively. Recombinant human TGF-1 and its antibody, which
neutralizes all subtypes of TGF-s, were from R&D System
(Minneapolis, MN) and Genzyme Diagnostics (Cambridge, MA), respectively.
Plasmids and cells
Zf9 mammalian (Zf9-pCIneo) and Drosophila
(Zf9-pAC) expression plasmids were constructed as previously
described.39 The uPA-luciferase expressing vector,
pGL2-2350, containing the human uPA promoter (2345 to 32), its
deletion mutant, pGL2-GC, containing the first 65 bp and GC box and
TATA box region (2345 to 2280 and 91 to +32,
respectively), and another deletion mutant, pGL2- GC, which contains
uPA promoter devoid of the GC box region (71 to 28), were
as described.36,37 Bovine aortic endothelial cells (BAECs)
were isolated and grown in minimal essential medium (MEM; GIBCO
BRL, Life Technologies, Rockville, MD) containing 10% fetal calf serum
(FCS). Drosophila S2 cells were generous gifts from Dr H. Tanaka, National Institute of Sericultural and Entomological Science,
Tsukuba, Japan and maintained in Shields and Sang M3 insect
medium (Sigma) containing 10% FCS.
Northern blotting
Isolation of total RNA from BAECs and its Northern blot analyses
were performed as described previously.54 RNA was separated through 1% agarose-formaldehyde gel electrophoresis and transferred onto the Biodyne nylon membranes (Pall Biosupport, New York, NY). Membranes were hybridized with 32P-labeled cDNA for either
human Zf9,39 bovine uPA,37 bovine uPAR,54 or human PAI-1,54 and rehybridized with
chicken glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal
control.54 Signals were quantitated using a Fujix BAS 2000 Bioimaging analyzer (Fuji Photo-Film, Tokyo, Japan).
Western blotting
Western blotting was performed as described previously37
using rabbit anti-Zf9 polyclonal antibody (final 1:10,000 dilution), and goat antirabbit IgG antibodies conjugated with peroxidase (Jackson
ImmunoResearch Laboratories, West Grove, PA). The signals were detected
with an Amersham-Pharmacia (Buckinghamshire, UK) ECL system. The blot
was reprobed with antirabbit GAPDH (Chemicon Int., Temecula, CA).
Gel shift assay
Rat zf9 cDNA was cloned into pGEX-3T (Pharmacia Biotech,
Sweden), and transformed in Escherichia coli BL21. Following
induction by 2 mM isopropyl -D-thiogalactoside for 4 hours, the cells were disrupted by sonication, and Zf9-glutathione
S-transferase (GST) fusion protein was purified using
glutathione-Sepharose (Amersham-Pharmacia) from cell homogenates.
Purified GST protein (Santa Cruz) was used as a control.
Oligonucleotides corresponding to 63 to 32 of the human
uPA promoter (wild-type uPA GC box),37 which contains 3 GC
boxes, as well as mutant oligos, in which mutations were made in 1 or
all of 3 GC boxes, were synthesized. Sequences of these
oligonucleotides are indicated in Figure 2A. Oligonucleotides were
double-stranded and labeled with  32-P adenosine
triphosphate (DuPont, Wilmington, DE) by polynucleotide kinase
(Boehringer Mannheim, Mannheim, Germany). For gel shift assays, 20 ng of Zf9-GST fusion protein was preincubated for 15 minutes
at 4°C with or without unlabeled oligonucleotide, then 3.5 ng
labeled oligonucleotide (10,000 µCi/mol) was added in the presence of
1 µg of dI-dC (Pharmacia) in 20 µL binding buffer (20 mM Hepes, pH
7.4, containing 1 mM MgCl2, 10 µM ZnSO4, 20 mM KCl, and 8% glycerol). The reaction mixture was
incubated for 15 minutes at 4°C and separated on a 6%
polyacrylamide gel. The gel was dried and exposed on films for a Fujix
BAS 2000 Bioimaging analyzer (Fuji Photo-Film).
Transient transfection
The BAEC cultures were plated on 35-mm dishes at 80% confluency.
Drosophila S2 cells were grown in 35-mm dishes
(5 × 106 cells/dish). Transient transfection was
performed using Lipofectamine Plus reagents (GIBCO, BRL, Life
Technologies Inc, Rockville, MD) in 1 mL serum-free medium
containing either Zf9-pCIneo or Zf9-pAC, with or
without 1 µg pGL2-2350, along with pRL-CMV (Renilla
luciferase, 100 ng/dish, Promega, Madison, WI) as an
internal standard to normalize transfection efficiency. pCIneo or pAC
was used as empty vector to adjust total amount of DNA transfected
always to 2 µg per each sample. After a 4-hour incubation, 1 mL of
medium containing 20% FCS was added to the cultures, and then further
incubated for 48 hours. Thereafter, either luciferase activity or
endogenous PA activity was determined in cell lysates, or total RNA was
isolated and used for Northern blotting.
Luciferase assay
Cells were harvested by scraping (BAECs) or by centrifugation (S2
cells) and lysed into 120 µL of the commercial lysis buffer (Promega). Luciferase activity was determined in 10 µL of lysate using a commercial kit (Promega) and luminometer (Turner Designs Instrument, Sunnyvale, CA). Transfection efficiency was
normalized by Renilla luciferase activity measured in the same
cell lysate at the same time.
Assay of cellular PA activity and membrane plasmin activity
Cellular PA activity was measured using the chromogenic substrate
S2403 as described previously and expressed as urokinase unit per
milligram of protein in the sample.37 Protein concentration was measured by bicinchoninic acid (BCA) (Pierce,
Rockford, IL) assay using bovine serum albumin (BSA) as the standard.
Plasmin bound to the cell surface was recovered with tranexamic acid
and assayed as described previously.54 This plasmin is
originally derived from plasminogen present in the serum.
Zf9 and uPA expression in vivo
The distal half of the left common carotid artery of a 10-week-old
Sprague-Dawley rat was denuded of endothelium by 3 passages of a 2-F
balloon catheter inserted through the left external carotid artery as
reported previously.55 Pentobarbital (intraperitoneal, 30 mg/kg) and 1% xylocaine (local) were used for anesthesia. Animals were
killed with an overdose of pentobarbital, and the artery was perfusion
fixed with 3% paraformaldehyde and excised. Paraffin-embedded serial
sections were stained using Vectastain ABC elite kit (Vector Laboratories, Burlingame, CA) according to the manufacturer's instructions with antibodies against rat Zf9 (final 1:500 dilution), rat uPA (final 1:50 dilution), or nonimmune rabbit serum (final 1:50
dilution). Color development was made with diaminobenzidine tetrahydrochloride and nuclear counterstain with hematoxylin. Procedures were examined and authorized by the institutional committee.
TGF- assays
Measurement of TGF- was performed using either a
bioassay (cellular PA assay with BAECs) or enzyme-linked immunosorbent
assays (ELISAs) specific for either TGF-1 or TGF-2 (Promega).
BAEC cultures were transfected with Zf9 expression vector and incubated for 36 hours. The cultures were then rinsed with phosphate-buffered saline (PBS) and further incubated with 1 mL of MEM containing 0.1%
BSA for an additional 12 hours to generate conditioned medium. Cellular
PA assays for TGF- were performed as described
previously.56 Briefly, following an 8-hour incubation of
test BAECs with each sample conditioned medium in the presence or
absence of 20 µg/mL either anti-TGF- antibody or nonimmune
antibody, cell lysate was prepared and PA activity levels of each cell
lysate were determined. The concentration of TGF- in the conditioned
medium was calculated from the decrease in PA levels by comparison with
a standard curve made with recombinant human TGF-1. The specificity
of the assays was verified by controls using anti-TGF- antibody. The
amount of total (active plus latent) TGF- was determined following
conversion of all latent TGF- to active TGF- by acidification of
the sample (pH 3, 1 hour at room temperature), followed by
neutralization. The lowest limit of this bioassay is about 2 pg/mL. For
ELISAs, conditioned medium was concentrated 10-fold on Centriprep
concentrator (Amicon Inc, Beverly, MA) and assayed for
either active TGF-1 or TGF-2 according to the manufacture's
directions, because both ELISAs detect a minimum of 25 pg/mL TGF-1
and 32 pg/mL TGF-2, respectively. Final concentrations were
calculated accounting for measured losses. The concentration of total
TGF-s was determined in unconcentrated medium immediately after acid
activation of all latent TGF-s.
Statistics
Significance was determined by the 2-tailed t test.
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Results |
Expression of Zf9 by BAECs
We first determined whether BAECs express Zf9. As seen in Figure
1A, BAECs maintained in serum-free medium
barely expressed a zf9 mRNA transcript of 4.5 kb (lane 1).
Incubation of the cells with 10% FCS-containing medium induced
zf9 mRNA expression in a time-dependent manner after 2 hours
(lanes 3-6). This induction was potentiated by inclusion of 10 ng/mL
PMA (lanes 7-11). Similar induction of Zf9 was also observed in protein
levels using Western blot (Figure 1B). PMA alone induced zf9
mRNA and protein expression in the absence of serum (data not shown).
When serum was removed after a 5-hour exposure in the absence of PMA,
Zf9 expression disappeared within 3 to 4 hours (Figure 1C and D,
lanes 1-5). On the other hand, the down-regulation of Zf9 by serum
withdrawal in the presence of PMA is only partial (Figure 1C and D,
lanes 6-10), because PMA can stimulate Zf9 production. These results indicate that Zf9 is a serum- and PMA-inducible factor expressed by
BAECs. Furthermore, PMA may slow the degradation of Zf9. None of the
following serum factors induced Zf9 when added individually to
serum-free medium: platelet-derived growth factor, TGF-, tumor necrosis factor-, and interleukin-1, or retinoic acid.
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| Fig 1.
Expression of Zf9 by BAECs.
(A) and (B) Time course in induction by serum and PMA. After a 5-hour
preincubation with serum-free medium confluent BAEC cultures in either
15-cm dishes or 6-cm dishes were incubated with 10% FCS-containing
medium for the indicated times, in the absence and presence of 10 ng/mL
PMA. Cell lysate was prepared from each 15-cm dish, total RNA was
isolated, and 30 µg of each RNA was used for detection of zf9
mRNA in Northern blotting (panel A). Cells on each 6-cm dish were
scraped into 60 µL of sodium dodecyl sulfate (SDS) sample buffer, and
rapidly passed 20 times through a 17-gauge needle to shear nucleic
acids. A portion (30 µL) of each cell lysate was immediately
subjected to SDS-polyacrylamide gel electrophoresis on 13% resolving
gel without reducing conditions, and changes in Zf9 protein levels were
analyzed by Western blotting as described in "Materials and
Methods" (panel B). Lane 1, control; lanes 2-6, without PMA; lanes
7-11, with PMA. Each experiment was repeated 5 times with similar
results and representative results are shown. (C) and (D) Disappearance
of Zf9 expression by BAECs after depletion of serum. Following a 5-hour
incubation with 10% serum ± 10 ng/mL PMA, BAECs were then
incubated with serum-free medium ± 10 ng/mL PMA, and harvested at
increasing intervals thereafter. The zf9 mRNA levels (panel C)
and Zf9 protein levels (panel D) were analyzed by Northern blotting and
Western blotting, respectively. Lanes 1-5, without PMA; lanes 6-10, with PMA. Each experiment was repeated 3 times with similar results and
representative results are shown. (E) Superinduction of Zf9 mRNA in the
presence of cycloheximide. After a preincubation in serum-free medium
for 5 hours, confluent BAEC cultures were incubated for 3 hours with
either fresh serum-free medium or 10% serum-containing medium in the
absence or presence of 6 µg/mL cycloheximide (CHX). Cell lysates were
prepared and the changes of zf9 mRNA levels were assessed by
Northern blotting. Lane 1, no stimulus; lane 2, serum alone; lane 3, cycloheximide alone; lane 4, serum plus cycloheximide. The experiment
was repeated 4 times with similar results and a representative result
is shown.
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Next, the effect of the protein synthesis inhibitor, cycloheximide, on
zf9 mRNA expression was assessed to determine if induction by
serum comprised an immediate-early response. Addition of 6 µg/mL
cycloheximide did not abrogate the induction of zf9 mRNA by
serum, but rather resulted in prominent superinduction of the transcript (Figure 1E, lanes 3 and 4), confirming the immediate-early nature of zf9 induction in BAECs.
Binding of recombinant Zf9 to the uPA GC box
To determine if Zf9 interacts with the uPA promoter sequences, we
examined binding of Zf9-GST to native and mutated GC boxes within the
uPA promoter by gel shift assay (Figure
2B). Zf9-GST fusion protein strongly bound
to the wild-type uPA GC box (lane 2), whereas none was detected for GST
alone (lane 4). Two shifted bands were detected. In experiments not
shown, the higher molecular weight band corresponds to intact Zf9 based
on Western blotting of the nondenaturing gel following transfer to
nylon, and a lower molecular weight band represents a degradation
product containing only the DNA binding domain.39
Interaction of both intact and degraded Zf9 with labeled
oligonucleotide was completely inhibited by a 20-fold excess of
unlabeled oligonucleotide (lane 3). Addition of anti-Zf9 antibody
abolished rather than supershifted the interaction between recombinant
Zf9 and the oligonucleotide (data not shown), similar to what we
reported previously using a consensus GC box oligonucleotide as a
probe.39
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| Fig 2.
Binding of Zf9-GST to GC boxes in the uPA promoter.
Binding of Zf9-GST fusion protein to the 32P-labeled uPA GC
box oligonucleotide was determined by gel shift assay. Four different
mutant oligonucleotides were tested, which contained mutations in one
or all of the GC boxes as shown. Sequences of these oligonucleotides
are indicated in panel A. Protein-DNA complexes were separated through
a 6% polyacrylamide gel and visualized on an image analyzer (panel B).
Lane 1, wild uPA GC box alone; lane 2, wild uPA GC box + Zf9-GST; lane
3, wild uPA GC box + Zf9-GST in the presence of a 20-fold excess of
unlabeled (cold) oligonucleotide; lane 4, wild uPA GC box + GST; lanes
5-8, mutant uPA GC boxes + Zf9-GST. Lane 5, oligonucleotide with
mutations in all 3 GC boxes (abbreviated as "All"); lane 6;
mutant oligonucleotide in which 5' site GC box was mutated
(abbreviated as "5'"); lane 7, mutant oligonucleotide in
which the middle GC box was mutated (abbreviated as "Mid"); lane
8; mutant oligonucleotide in which 3' site GC box was mutated
(abbreviated as "3'"). The experiment was repeated 3 times
with similar results in all experiments; representative results are
shown.
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We determined which GC boxes among 3 uPA promoter GC boxes are critical
for Zf9 binding. Intact Zf9 did not bind to the oligonucleotides containing mutations in the middle GC box (lanes 5 and 7), but did bind
to the mutants preserving the middle GC box (lanes 6 and 8), indicating
that this middle GC box is the most critical for interaction between
Zf9 and the uPA promoter.
Enhancement of PA activity by Zf9 via transactivation of
uPA gene
Transactivation of uPA by Zf9 was explored using transient
transfection. Cotransfection of a uPA reporter (pGL2-2350) and the Zf9
expression vector (Zf9-pCIneo) in BAECs resulted in a concentration-dependent enhancement of luciferase activity (Figure 3A). Relative luciferase activity increased
maximally to 6-fold compared to the control cells transfected only with
pCIneo empty vector. To confirm that transactivation of uPA by Zf9 was
not dependent on coexpression of Sp1, the same analysis was performed in Drosophila S2 cells, which are devoid of endogenous Zf9 as well as Sp1-like activity.35,39 A similar effect was
obtained and interestingly, the relative activity was greater in this
cell type, probably due to low basal activity (Figure 3B). In Figure 3C
we compared the effect of Zf9 expression on transactivation of
wild-type uPA promoter (pUK-Luc) or its mutants, either
containing only GC and TATA boxes (pUK GC-Luc) or deficient in
GC boxes (pUK GC-Luc). The basal activities of
pUK GC-Luc (sample 3) and pUK GC-Luc (sample
5) were, respectively, 360% and 13% of that of the wild-type
pUK-Luc (sample 1), suggesting that the GC box region is
necessary for full transactivation of the uPA gene as reported previously.36 Zf9 transfection enhanced transactivation of
both wild-type pUK-Luc and pUK GC-Luc to the same
extent (5.4~5.7-fold), but not of pUK GC-Luc,
indicating that transactivating activity of Zf9 requires GC boxes
within uPA promoter.
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| Fig 3.
Transactivation of the uPA promoter by Zf9 in BAECs and
Drosophila S2 cells.
(A) and (B) BAEC and Drosophila S2 cell cultures grown on 35-mm
dishes were cotransfected with a combination of the indicated amounts
of Zf9 expression vector (Zf9-pCIneo or Zf9-pAC) and 1 µg of pGL2-2350, luciferase reporter gene fused with the uPA
promoter, as described in "Materials and Methods." After a
48-hour incubation, cell lysates were prepared, luciferase activity in
each lysate was determined and expressed as fold increase. Panel A,
BAECs; panel B, Drosophila S2 cells. Each value represents the
average ± SD from triplicate determinations. Each experiment was
repeated 3 times with similar results and representative results are
shown. (C) Transactivation by Zf9 of the uPA promoter via GC box
regions. BAEC cultures were cotransfected with a combination of 500 ng
each of either pCIneo or Zf9-pCIneo plus 1 µg each of either
pGL2-2350 (pUK-Luc), pGL2-GC (pUK GC-Luc), or pGL2-GC (pUK
GC-Luc). Cell lysates were prepared, and luciferase activity of each
lysate was determined. Data are expressed as relative luciferase
activity compared to the activity of pUK-Luc cotransfected with pCIneo
alone. The numbers in parentheses to the right of each bar indicate
fold-induction calculated for each reporter. Samples 1 and 2, pUK-Luc;
samples 3 and 4, pUK GC-Luc; samples 5 and 6, pUK GC-Luc. Odd
numbers, pCIneo; even numbers, Zf9-pCIneo. Each value
represents the average ± SD from triplicate determinations.
Experiment was repeated 3 times with similar results and representative
results are shown.
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We next examined the effect of transiently transfected Zf9 on
endogenous uPA expression and activity in BAEC cultures. uPA mRNA
expression in BAEC cultures was up-regulated 200% as determined by
densitometry normalized to GAPDH expression following transfection of
zf9 cDNA despite the typically low (< 20%)
efficiency of transient transfection (Figure
4A, upper bands). Because both uPAR and
PAI-1 promoters also contain GC boxes,57,58 we examined the
effect of Zf9 on these mRNAs. In contrast to uPA, mRNA expression of these genes was not affected by Zf9 (Figure 4A, second and third bands). The selective increase in uPA mRNA but not its inhibitor or
receptor suggested that Zf9 might enhance net PA activity. Indeed,
transient transfection with Zf9 increased endogenous cellular PA
activity to 200% (Figure 4B). Furthermore, membrane-associated plasmin
levels were increased from 9.8 ng/106 cells to 27.5 ng/106 cells after transfection with Zf9 for 48 hours.
These results suggest that Zf9 promotes uPA transcription through GC
box region and enhances surface plasmin levels of BAEC cultures.
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| Fig 4.
Effect of Zf9 on endogenous PA activity in BAEC cultures.
(A) Effect on mRNA expression of uPA, uPAR, and PAI-1. BAEC cultures
grown on 10-cm dishes were transfected with 8 µg of
Zf9-pCIneo. As a control, cells were transfected with the same
amount of pCIneo. Forty-eight hours after transfection, cell lysates
were prepared, total RNA was extracted, and mRNA expression of uPA,
uPAR, PAI-1, and GAPDH in each sample was assessed by Northern
blotting. Radioactivity of each band was detected on an image analyzer,
and relative intensity of each band was quantitated, normalized against
the intensity of GAPDH, and expressed as arbitrary units in parentheses
under each band. Each experiment was repeated 3 times with similar
results in all, and representative results are shown. (B) Effect on
BAEC PA activity levels. BAEC cultures grown on 35-mm dishes were
transfected with the indicated amounts of Zf9-pCIneo.
Forty-eight hours after transfection, cells were harvested in lysis
buffer, and PA levels in each lysate were determined using chromogenic
substrate, S-2403, and expressed as urokinase (UK) unit (U)/mg of
protein in the sample. Each value represents the average ± SD from
triplicate determinations. Points marked by an asterisk differ
significantly (P < 0.05) from control cells transfected
with 2 µg of pCIneo alone. Experiment was repeated 4 times with
similar results in all, and representative results are shown.
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Coordinated expression of Zf9 and uPA proteins by vascular
endothelial cells of the carotid artery and aorta after balloon
injury
Vascular endothelial cells express a small amount or no uPA in
normal arteries, but uPA expression dramatically increases after
vascular injury.12,55 To study the role of Zf9 in response to vascular injury in vivo, we injured the distal half of the left
carotid artery of rats with a balloon catheter and immunostained carotid artery with Zf9 and uPA antibodies at various time points after
the injury. At 3 hours after the injury, neither Zf9 nor uPA
immunoreactivity was detected in the vascular endothelial cells or in
the smooth muscle cells (Figure 5, panels A
and B, respectively). Within 2 days of injury, however, both Zf9 and uPA immunoreactivities were detected in the vascular endothelial cells
(down arrows in Figure 5, panels D and E, respectively), whereas
neither was detected in the endothelial cells of uninjured control
rats. Weak immunoreactivity was also detected in the medial smooth
muscle cells of the injured carotid artery (up arrows). Interestingly,
Zf9 was induced not only in endothelial cells near the wound edge, but
also in the adjacent proximal half of carotid artery, as well as in the
endothelial cells of the aorta in the same animal. Nonimmune serum did
not give any positive staining (Figure 5, panels C and F).
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| Fig 5.
Concomitant expression of Zf9 and uPA in arterial
endothelial cells after carotid balloon injury in rats.
The distal half of the left carotid artery of rats was injured with a
balloon catheter. At 3 hours and 2 days after injury, the carotid
artery was perfusion fixed with 3% paraformaldehyde, excised, fixed
again with 3% paraformaldehyde, and paraffin embedded. Serial sections
were stained with anti-Zf9 antibody (panels A and D), anti-uPA antibody
(panels B and E) or nonimmune antibody (panels C and F) as described in
the Materials and Methods. Panels A through F show the typical staining
pattern of the adjacent proximal half of carotid artery at 3 hours
(panels A-C) and 2 days (panels D-F) after injury. Down arrows indicate
the endothelial cells. Up arrows indicate the smooth muscle cells.
Scale bar, 50 µm.
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Induction of TGF- by Zf9 via enhancement of PA activity
Finally, we examined whether Zf9 has biologic activity in this
system via enhancement of PA/plasmin activity. We speculated that
up-regulation of PA/plasmin by Zf9 might result in the activation of
latent TGF- as has been demonstrated in other
systems.25,59,60 This hypothesis was tested by 2 different
assays. First, as determined by bioassays, we observed that transient
transfection of Zf9 cDNA induced the generation of active TGF- in
BAEC cultures during a subsequent 12-hour incubation with serum-free
medium (Figure 6A, sample 3). This increase
was eliminated by inclusion of a plasmin inhibitor, aprotinin, during
the preparation of the conditioned medium (Figure 6A, sample 4),
suggesting that TGF- formation resulted from
proteolytic activation of latent
TGF-.17-25,59,60 On the other hand, total
TGF- levels only moderately (35%) increased (Figure
6B, sample 3), and there were no statistically significant differences
between each sample. A similar result was also obtained with TGF-1
and TGF-2 ELISAs, although the basal levels of active TGF-s (sum
of TGF-1 and TGF-2 concentrations) were higher than those
determined with the bioassay (Table 1).
Transient transfection of Zf9 did not alter endogenous TGF-1 and
TGF-2 mRNA levels in BAEC cultures (data not shown). These data
indicate that Zf9 can induce TGF- activity in BAECs and that the
increased TGF- in this system derived mainly from conversion of the
latent to the active form of the cytokine, due at least in part to
increased fibrinolytic activity.
View larger version (21K):
[in this window]
[in a new window]
| Fig 6.
Activation of latent TGF- following transfection of
Zf9.
BAEC cultures grown on 35-mm dishes were transfected with 1 µg of
pCIneo or Zf9-pCIneo as described above. After a 36-hour
incubation, medium was changed to serum-free MEM containing 0.1%
BSA, and cells were further incubated for 12 hours in the absence and
presence of 50 µg/mL aprotinin (Apr). The concentration of active
(panel A) and total (panel B) TGF- in each conditioned medium was
determined by the bioassays as described in "Materials and
Methods." Each value represents the average ± SD from triplicate
determinations. Each experiment was repeated 3 times with similar
results in all, and representative results are shown. Sample 1, pCIneo;
sample 2, pCIneo with inclusion of aprotinin; sample 3, Zf9-pCIneo; sample 4, Zf9-pCIneo with inclusion of
aprotinin. Points marked by an asterisk differ significantly
(P < 0.05) from the control (sample 1).
|
|
| |
Discussion |
We have demonstrated a potential role of Zf9 in regulating vascular
injury through its marked stimulatory effects on uPA gene expression and TGF- activity. Cultured BAECs express Zf9 in response to serum as well as PMA (see Figure 1A), which is a model agonist of a
hitherto known early response gene, Egr-133; we
have not yet identified the responsible serum factor. The kinetic
analysis of zf9 mRNA and protein (see Figures 1A-D) suggests
that both are labile, and therefore withdrawal of appropriate stimulus
culminates in their rapid disappearance. This observation, combined
with superinduction by cycloheximide (see Figure 1E), supports the role
of Zf9 as an immediate-early gene, as previously observed in hepatic
stellate cells in vivo.38 As shown in Figure 1B and D, Zf9
protein is expressed by BAECs as a doublet of 46 kd and 42 kd,
consistent with our previous finding in hepatic stellate cells.39 Furthermore, transient transfection of human or
rat Zf9 into BAECs also yielded the same doublet in Western blot (data not shown). As we have suggested previously, the 14-kd or 10-kd size
differences from 32 kd, which is the predicted size of the unmodified
polypeptide, may be explained by posttranslational modifications, but
this has not yet been tested directly.39
We have also demonstrated that both the interaction between recombinant
Zf9 and the uPA promoter (see Figure 2) and transactivation of the
uPA gene by Zf9 (see Figure 3) are dependent on GC boxes within
the promoter in vitro. Incubation of Zf9-GST with mutated promoter
sequences establishes that the middle GC box is the most essential for
Zf9 binding. The substantial increase in endogenous uPA activity after
transient transfection of Zf9 (see Figure 4), despite a typical
transfection efficiency of only 10% to 20%, underscores the potency
of this factor in up-regulating uPA expression. Importantly, vascular
injury caused by a balloon catheter induces the expression of Zf9 by
endothelial cells in vivo (see Figure 5). Concomitant induction of uPA
in this tissue underscores the notion that Zf9 may play an important
role in the uPA gene expression induced by vascular injury.
This finding supports our in vitro observations and agrees with
findings of previous investigators characterizing uPA induction after
vascular injury in vivo.12 We acknowledge, however, that
the current data do not establish a direct role of Zf9 in uPA
expression in vivo.
Zf9 belongs to an enlarging family of zinc finger Krüppel-like
transcription factors.42-52 The physiologic roles and
distribution of these factors are distinct.52 Sp1
is widely expressed in various types of cells at a fairly constant
level and is involved in basal transactivation of many genes including
uPA.36,37 Zf9 is also widely expressed but is an
early response gene in both hepatic stellate38,39 and
vascular endothelial cells (current study) during the wound healing
response. In this context, Zf9 resembles Egr-1, a
non-Krüppel-like zinc finger transcription factor that has been
identified also as an early gene induced by serum, PMA, and tissue
injury, and can substitute for Sp1 in promoting transcription of genes
including uPA.33 Therefore, it is of great interest to know
how Sp1, Egr-1, Zf9, and other Krüppel-like factors interact in
regulating uPA expression. In preliminary studies we have documented a
synergism and physical interaction between Zf9 and Sp1.61
This contrasts with the interplay of Sp1 and Egr-1.33
We previously demonstrated that Zf9 transactivates promoters of
collagen 1(I), TGF-1, and its signaling receptors in hepatic stellate cells.39,53 Activation of latent TGF- via uPA
induction provides an additional mechanism through which TGF-1
activity is augmented by Zf9 (see Figure 6 and Table 1). This has also been observed in other systems in which uPA is up-regulated, for example, in response to basic fibroblast growth factor or retinoic acid.59,60 Activation of latent TGF- has been implicated
as an important regulator of vascular smooth muscle cell
differentiation, endothelial cell quiescence, and extracellular matrix
production during vessel morphogenesis. It seems that latent TGF- is
activated through different mechanisms under physiologic and pathologic situations. It is intriguing to determine if Zf9 might also stimulate the expression of other TGF- activators, such as thrombospondin and
integrin, v6.62,63 We speculate that Zf9 may promote different GC box-containing gene promoters in a tissue and cell context-dependent manner. For example, the current (see Figure 3B) and
previous39,53 data suggest that in S2 cells Zf9
transactivates the SV40 promoter as well as promoters of TGF-1 and
uPA, but not promoters of collagen 1(I), types I and II TGF-
receptors. Although the percentage of active TGF- in our system was
low (2.4% in Figure 6 and 4.3%-6.3% in Table 1), 40 to 60 pg/mL
TGF- is enough to exert its biologic effects on BAECs as we and
others have reported.23,56,59 We cannot explain the reason
for the differences in the concentration of active TGF- determined
by the biologic assays (see Figure 6) and ELISAs (see Table 1). We
speculate that artificial activation might occur during concentration of the conditioned medium prior to ELISA, resulting in high basal levels. It would be difficult to evaluate if serum-induced Zf9 also
generates active TGF- in BAEC cultures because 10% serum used in
the current study already contains 99.5 ± 4.7 pg/mL active TGF-1 and 11.2 ± 3.8 pg/mL active TGF-2 as measured by
ELISA. This preexisting TGF- might counteract the ability of
Zf9 to enhance uPA expression, inferring that the system
is self-regulated, similar to what has been reported
previously in other systems.64
In summary, we have shown that both in vitro and in vivo Zf9
is expressed by vascular endothelial cells in response to certain stimuli and can promote transactivation of uPA, which leads to activation of latent TGF-, suggesting that this system may have an
important role in vascular injury response. The present data and
previous findings25,38,39 strongly suggest that rapid, early induction of Zf9 may promote fibrogenesis by potentiating the
proteolytic activation of latent TGF- in hepatic stellate cells. It
will be important to determine if Zf9 regulates uPA activity and
TGF-1 responses in other in vivo models of tissue injury including
atherosclerosis and restenosis,6,20 cutaneous wound
healing,2 pulmonary intestinal pneumonia and
fibrosis,22,63 and hepatic fibrosis.1,39
| |
Acknowledgments |
We gratefully acknowledge F. Blasi for helpful advice
and critical reading of the manuscript; H. Tanaka, W.-D. Schleuning, and D. J. Loskutoff for Drosophila S2 cells and constructs; and L. Wong and C. Iijima for their technical assistance.
| |
Footnotes |
Submitted March 19, 1999; accepted October 5, 1999.
Supported partly by Grant-in-Aids from the Ministry of Education,
Science, Sports and Culture 10780395, Grant for "Biodesign Research
Program" and "Multibioprobe Research Program" from RIKEN, The
Special Coordination Funds for Promoting Science and Technology from
the Science and Technology Agency, and the National Institutes of
Health (DK37340).
S.K. and S.H. contributed equally to this work.
Reprints: Soichi Kojima, Laboratory of Molecular Cell
Sciences, Tsukuba Life Science Center, The Institute of Physical and
Chemical Research (RIKEN), Koyadai, Tsukuba, Ibaraki 305-0074, Japan;
e-mail: kojima{at}rtc.riken.go.jp.
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.
| |
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Top
Abstract
Introduction