Department of Medicine, Hematology and Oncology, University of Münster, Albert-Schweitzer Str. 33, D-48129 Münster, Germany
* Author for correspondence (e-mail: kienast{at}uni-muenster.de
Accepted 4 February 2002
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Summary |
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Key words: Urokinase, Growth factors, Smooth muscle cells, Proliferation, Atherogenesis
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Introduction |
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Growth factors and cytokines that regulate cell proliferation have been
shown to promote UPA production in various tumoral and normal cell-types
(Stopell et al., 1986;
Dong-Le Bourhis et al., 1998
;
Sieuwerts et al., 1999
;
Fibbi et al., 1999
;
Kitange et al., 1999
). In
addition, there is growing evidence of an association between UPA expression
and the mitogenic activity of the cells
(Grimaldi et al., 1986
;
Zavizion et al., 1998
;
Hibino et al., 1999
;
Scott et al., 1987
). Thus,
expression of UPA could be a key step for the regulation of cell proliferation
in response to growth factors. Indeed, in vitro studies have shown that UPA
acts as an autocrine mitogen in human melanoma cells
(Kirchheimer et al., 1989
) and
that growth-factor-induced proliferation of human hepatic cells is grossly
dependent on UPA (Fibbi et al.,
1999
). In addition, exogenous UPA induces proliferation and growth
of cells of various origins (Fibbi et al.,
1999
; De Petro et al.,
1994
; Koopman et al.,
1998
; Fischer et al.,
1998
: Fishmann et al., 1999) and recent studies have demonstrated
that UPA can also elicit a mitogenic response in human vascular SMC
(Kanse et al., 1997
;
Stepanova et al., 1999
;
Dumler et al., 1999
). However,
as yet it remains unclear whether this mitogenic response depends on the
proteolytic activity of UPA or requires binding to its cellular receptor
CD87.
Atherosclerotic and restenotic lesions are characterized by overexpression
of growth factors and their receptors
(Ross, 1993). Both,
platelet-derived growth factor (PDGF) and basic fibroblast growth factor
(bFGF) are known to play an important role in vascular SMC proliferation
(Ross et al., 1986
;
Lindner et al., 1991
). bFGF
has been found to upregulate UPA-expression in human and murine vascular SMC
both at mRNA and protein levels (Wang et
al., 1997
; Herbert et al.,
1997
). Although a similar effect for PDGF on SMC could not be
shown in the latter study (Herbert et al.,
1997
), PDGF has been reported to increase the expression of UPA in
human hepatic cells (Fibbi et al.,
1999
).
Regardless of the close relationship between growth factors and UPA during
cell proliferation, only one report in transgenic mice has dealt with the role
of endogenous UPA on the PDGF-and bFGF-induced growth of vascular SMC
(Herbert et al., 1997).
Therefore, the present study was undertaken to investigate whether endogenous
UPA contributes to the mitogenic response of human vascular SMC to PDGF and
bFGF, and to characterize the structural and functional properties of UPA that
are involved in its growth factor-like function.
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Materials and Methods |
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Cell culture
Human vascular SMC were obtained as confluent monolayers in passage two
from Technoclone (Austria). According to the manufacturer, cells were
delivered as a pool of SMC isolated from the umbilical veins of 10 donors by
the explant technique. SMC were identified by their characteristic `hill and
valley' growth appearance and by immunostaining for SMC -actin. Cells,
routinely used from the third to the seventh passage, were grown in DMEM
supplemented with 10% (v/v) heat inactivated FCS, 100 IU/ml penicillin, 100
µg/ml streptomycin and 2.5 µg/ml amphotericin B at 37°C in a humid
atmosphere of 5% CO2 in air. Cells were passaged using 0.25%
trypsin/0.02% EDTA (ethylenediaminetetraacetic acid). Culture medium was
changed every 2-3 days and a confluent SMC monolayer was obtained after about
7 days.
DNA synthesis
Vascular SMC were sparsely plated into 96-well plates
(1x103 cells/well) in the presence of 10% FCS in DMEM/Ham's
F12. At 24 hours, cells were washed and rendered quiescent with 0.5% FCS for
further 72 hours. After this period, proliferation was reinitiated with 10%
FCS or with 1% FCS in the presence of either PDGF (20 ng/ml) or bFGF (10
ng/ml) without and with anti-UPA-antibodies, anti-UPAR-antibodies,
p-aminobenzamidine (UPA-inhibitor), lysine analogues (-aminocaproic
acid, tranexamic acid), or the plasmin inhibitor aprotinin. After 24 hours,
5-bromo-2-deoxyuridine (BrdU, 10 µM, Roche Diagnostics) was added, and the
cultures were maintained for a further 24 hours. In other experiments, cells
were labeled with BrdU for 24 hours starting at time of stimulation with or
without PDGF and bFGF, with increasing concentrations (0-40 µM) of TBB or
DRB (protein kinase CK2 inhibitors) in the absence or presence of
p-aminobenzamidine or purified HMW-UPA. Incorporation of BrdU in the genomic
DNA of proliferating cells was measured by a colorimetric immunoassay
according to the manufacturer's instructions (Cell proliferation ELISA; Roche
Diagnostics). In a separate set of experiments, the vascular SMC were exposed
to BrdU for serial pulses of 6 or 12 hours each. The mitogenic activity of the
cells was expressed by the BrdU-labeling index (BrdU-LI), which was calculated
as follows:
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Cell proliferation
Vascular SMC growth rates were measured by plating
1x104cells/well in 24-well plates. Growth-arrested cells (as
described above) were stimulated with 10% FCS or with 1% FCS in the presence
of either PDGF (20 ng/ml) or bFGF (10 ng/ml) with and without
anti-UPA-antibodies or the UPA-inhibitor p-aminobenzamidine. Cells were
maintained in culture for a period of 7 days with medium changes every 48
hours. At 24 hour intervals, cells were trypsinized and counted under the
microscope in a Neubauer hemocytometer. Cell viability was monitored by Trypan
Blue dye exclusion.
Preparation of SMC-conditioned medium (SMC-CM) and cell extracts
To evaluate PDGF- and bFGF-induced expression of UPA, SMC were cultured in
24-well cluster plates and treated with 1% FCS in the absence or presence of
PDGF or bFGF, as described above, for time periods of up to 48 hours. After 24
and 48 hours, SMC-CM was collected, centrifuged at 3000 g for 5
minutes for removal of cell debris, and stored at -80°C until the assays
were performed. For UPA antigen extraction, the cells were washed with
phosphate-buffered saline (PBS) and harvested at 4°C using an acid
acetate/Triton X-100 buffer (Padró
et al., 1995). After two freeze-thawing cycles to break the cell
membranes, samples were further incubated for 15 minutes at 4°C and
finally centrifuged for 10 minutes at 5000 g. Supernatants were
stored at -80°C until analysis. The protein content in cell extracts was
determined by the bicinchoninic acid method using a commercial assay (Pierce
Chemical, USA). To evaluate the levels of UPA mRNA in SMC, total cellular RNA
was isolated using the guanidine isothiocyanate/phenol method
(Chomczynski and Sacchi,
1987
).
Reverse transcription-polymerase chain reaction (RT-PCR)
analyses
For semi-quantitative UPA messenger RNA (mRNA) analysis, 300 ng total RNA
was reverse transcribed. Complementary DNA (cDNA) was synthesized for 1 hour
at 37°C using random hexamers and moloney murine leukemia virus (M-MLV)
reverse transcriptase (Promega, Germany). UPA transcripts were amplified using
Taq polymerase (Promega) on a Hybaid thermocycler (MWG-Biotech, Germany) for
35 cycles (45 seconds at 94°C, 45 seconds at 58°C, 1 minute at
72°C and 5 minutes at 72°C). The following primer pairs were used for
the amplification of human UPA cDNA: 5'-TCCCGGACTATACAGACCAT-3'
(sense) and 5'-TCTCTTCCTTGGTGTGACTG-3' (antisense)
(Iwasaka et al., 1996). In all
PCR amplifications, glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 347 base
pairs) was co-amplified as an internal control for RNA integrity and
quantification, using the GAPDH primers 5'-CCCTCCAAAATCAAGTGGGG-3'
(sense) and 5'-CGCCACAGTTTCC-CGGAGGG-3' (antisense). The amplified
PCR products were separated on 6% polyacrylamide gels and stained with
ethidium bromide. For the estimation of UPA expression, the corresponding
signals were normalized against GAPDH using a fluorometric analysis system
(Gel Doc 1000; Bio-Rad Laboratories, Germany).
Quantification of UPA
UPA antigen was quantitatively determined in cell extracts and SMC-CM by a
commercial ELISA (Tintelize, Biopool, Sweden). Functional UPA in SMC-CM
(pro-UPA and two-chain active UPA) was measured by a biologic immunoassay
(Chromolize, Biopool, Sweden). Calibration curves were prepared by dilution of
UPA standard provided by the manufacturer either in culture medium or
extraction buffer as required. UPA-activity was directly evaluated in a
chromogenic assay using the substrate S-2444, as described by the providers
(Chromogenix, Sweden).
Statistics
Data are presented as individual data plots or as medians and interquartile
ranges (IQR). The statistical significance of overall differences between
groups was analyzed by the Kruskal-Wallis one-way analysis of variance. If the
test was significant, pairwise comparisons were done by the
multiple-comparisons' criterion. Differences between two independent groups
were analyzed by the Mann-Whitney rank sum test. The Wilcoxon matched-pair
signed rank test was used for comparison of differences within pairs. A
P value of 0.05 or less was considered significant.
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Results |
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|
|
Expression of UPA in vascular SMC after PDGF and bFGF
stimulation
Quiescent human vascular SMC constitutively expressed UPA. Median
concentrations of UPA antigen in cell lysates of growth-arrested cells did not
vary significantly during culture for periods of up to 48 hours [0 hours: 12.0
(IQR, 10.9-21.3) ng/mg extracted protein; 24 hours: 18.9 (11.3-22.2) ng/mg
extracted protein; 48 hours: 13.3 (11.2-17.3) ng/mg extracted protein].
To study the effects of PDGF and bFGF on UPA expression in human vascular SMC, growth-arrested cells were exposed to 1% FCS without (control) and with growth factors for periods of up to 48 hours as described in Materials and Methods. The steady-state levels of UPA antigen were not significantly altered when cells were treated with 1% FCS alone with median concentrations in cell lysates of 12.1 (7.1-19.1) ng/mg extracted protein after 24 hours and 10.8 (8.1-14.4) ng/mg extracted protein after 48 hours (Fig. 2A). In contrast, median UPA antigen levels in PDGF-stimulated SMC increased 1.9-times (IQR; 1.5-2.8) and 3.1-times (IQR; 2.7-4.4) over controls (1% FCS) after 24 and 48 hours in culture, respectively (P<0.01 for both time periods; Fig. 2A). More marked stimulation effects, although with a similar trend, were observed when human vascular SMC were treated with bFGF for up to 48 hours [median fold increase over controls: 24 hours, x3.7 (IQR; 2.5-7.3), P<0.001; 48 hours, x5.1 (IQR; 2.8-7.0), P<0.001, Fig. 2A].
|
Semiquantitative RT-PCR showed an increase in UPA transcripts in vascular SMC after exposure to PDGF and bFGF for 24 and 48 hours, respectively, which indicated PDGF- and bFGF-induced stimulation of UPA gene expression (Fig. 2B).
A similar trend as that described for UPA in cell lysates emerged for levels of UPA antigen in conditioned media. Control SMC (1% FCS) had consistently low levels of UPA antigen in culture supernatants over the 48 hour incubation period. After 48 hours in culture, UPA antigen in the conditioned media of growth factor stimulated vascular SMC increased by 7- to 11-times compared with the control SMC (Table 2). Functional UPA levels were measured by a biologic immunoassay that detects both active two-chain UPA and pro-UPA. We found that the pro-UPA form almost quantitatively accounted for total functional UPA in the conditioned media of both the control and the growth-factor-stimulated SMC (data not shown).
|
Effect of polyclonal antibodies against UPA on DNA synthesis of PDGF
and bFGF-stimulated vascular SMC
Since PDGF- and bFGF-induced UPA expression in SMC paralleled the induction
of DNA-synthesis, we next investigated whether endogenous UPA was involved in
the mitogenic response to these growth factors.
To this end, growth-arrested SMC were exposed to polyclonal goat anti-UPA IgG (398, American Diagnostica; 40 µg/ml) or goat non-immune IgG (40 µg/ml) for 15 minutes. Thereafter, cells were stimulated with PDGF or bFGF in the continuous presence of the neutralizing antibodies or the non-immune IgG for periods of up to 48 hours. In a first set of experiments, DNA-synthesis was analyzed by BrdU-uptake during the second 24 hour incubation period. Fig. 3 shows that in PDGF and bFGF stimulated SMC, the BrdU labeling-index (BrdU-LI, see Materials and Methods) reached median values of 0.86 (IQR, 0.75-1.10) and 0.70 (IQR, 0.57-0.75), respectively, compared with 0.26 (IQR, 0.20-0.33) in the control cells treated with 1% FCS alone (P<0.001 both for PDGF and bFGF plus vehicle, respectively, versus controls; Mann-Whitney test). In the presence of polyclonal anti-UPA IgG, there was a significant reduction in the BrdU-uptake induced by either PDGF (BrdU-LI: 0.65; P=0.004) or bFGF (BrdU-LI: 0.52; P=0.004). The BrdU-LI in growth factor stimulated SMC was not significantly affected by the presence of non-immune goat IgG (PDGF, 0.86; bFGF, 0.77).
|
We further examined the effect of the polyclonal antibody 398 (anti-UPA IgG) on BrdU-incorporation by vascular SMC through the time period (12-24 hours) when quiescent cells stimulated with PDGF and bFGF entered S phase (Table 1). Polyclonal anti-UPA IgG consistently reduced the BrdU-labeling index during both the 12-18 hour and the 18-24 hour intervals of stimulation with either PDGF (P=0.03 for both time periods) or bFGF (P=0.03 for both time periods) (Fig. 4A,B).
|
DNA synthesis in PDGF- or bFGF-stimulated vascular SMC is dependent
on the UPA-catalytic domain but does not require UPA-UPAR interaction
In order to characterize the structural and functional properties of UPA
that are involved in PDGF- and bFGF-induced DNA synthesis, we examined the
effects of a specific monoclonal antibody against the UPA-B chain blocking the
catalytic site and enzymatic activity (mAb 394OA, American Diagnostica) and a
monoclonal antibody against the UPA-A chain that blocks UPAR binding (mAb
3921, American Diagnostica). As described above, vascular SMC were stimulated
with the growth factors in the absence or in the continuous presence of the
monoclonal antibodies (40 µg/ml). Fig.
5 shows that median PDGF- and bFGF-induced DNA synthesis decreased
by 70% (P<0.001) and 89% (P<0.001), respectively, in
the presence of mAb 394OA. In contrast, BrdU-uptake was not significantly
affected by mAb 3921 [BrdU-LI, PDGF: 0.91 (0.83-0.94) versus 0.96 (0.90-1.03)
in controls; bFGF: 0.74 (0.71-0.79) versus 0.83 (0.80-0.85) in controls]
(Fig. 5). In addition, a
monoclonal antibody directed against the UPAR (mAb 3936, American Diagnostica;
Fig. 5), which interferes with
UPA binding, was also ineffective in preventing BrdU-incorporation into DNA of
human vascular SMC stimulated with either PDGF [BrdU-LI: 0.89 (0.86-0.94)] or
bFGF [BrdU-LI: 0.79 (0.68-0.80)].
|
In a subsequent series of experiments, BrdU-uptake in PDGF and bFGF
stimulated vascular SMC was evaluated in the presence or absence of the
UPA-inhibitor p-aminobenzamidine (Geratz
and Cheng, 1975). Preliminary experiments demonstrated that
p-aminobenzamidine inhibited UPA protease activity in a dose-dependent manner
(15-1000 µM; evaluation by a chromogenic assay), with a
Kd of 180 µM (data not shown). The presence of
p-aminobenzamidine significantly reduced the PDGF- and bFGF-induced DNA
synthesis in cultured SMC with a dose-dependent effect between 50 µM and
1000 µM (PDGF, 19-84% reduction; bFGF, 11-94% reduction;
Fig. 6).
|
Role of the UPA catalytic domain on vascular SMC cell proliferation
induced by PDGF and bFGF
The relevance of the catalytic domain of UPA for the mitogenic effects of
PDGF and bFGF on human vascular SMC was also studied by cell growth-curves.
SMC stimulated with PDGF or bFGF had median doubling times of 47 hours and 50
hours, respectively, and the median cell number increased 12-fold after 7 days
of culture in the presence of PDGF (Fig.
7A) and 10-fold when cultured in the presence of bFGF
(Fig. 7B). With regard to
PDGF-induced SMC proliferation, doubling times were 1.2-times prolonged in the
presence of polyclonal anti-UPA IgG (40 µg/ml; P=0.05), 2.3-times
in the presence of mAb 394OA (40 µg/ml; P=0.03), and 1.4-times in
the presence of p-aminobenzamidine (250 µM; P=0.03). In accordance
with these findings, median cell numbers after 7 days in culture were reduced
by 35%, 74% and 52%, respectively (P<0.01 for all treatments).
Similarly, bFGF-stimulated SMC had longer doubling times in the presence of
polyclonal anti-UPA IgG (x1.4, P=0.05), mAb 394OA (x2.8,
P=0.03) and p-aminobenzamidine (x1.3, P=0.03). Median
cell numbers were reduced by 50%, 79% and 41% (P<0.01 for all
treatments) compared with the controls (bFGF alone) after 7 days in culture.
Non-immune-goat IgG, non-immune-mouse-IgG and the vehicle of
p-aminobenzamidine did not affect PDGF- or bFGF-induced cell growth (data not
shown).
|
Role of plasmin generation in the UPA mediated PDGF- or bFGF-induced
DNA synthesis in vascular SMC
To examine whether plasmin generation is required for the
catalytic-domain-dependent mediator function of UPA in PDGF- and bFGF-induced
SMC proliferation, the effects of the plasmin inhibitor aprotinin and of the
lysine analogues tranexamic acid and -aminocaproic acid were tested.
Lysine analogues have been shown to interfere with the binding of plasminogen
to the cell surface and, therefore, prevent the generation of plasmin
(Dunn and Goa, 1999
; Vldavsky,
1987). Table 3 shows that the
addition of 10 mM tranexamic acid, 10 mM
-aminocaproic or 100 µM
aprotinin to the culture medium had no effect on DNA synthesis, indicating
that the UPA-mediated mitogenic activity of PDGF and bFGF did not occur
through the generation of plasmin.
|
Simultaneous inhibition of UPA and CK2 activity reduced PDGF- or
bFGF-induced DNA synthesis in vascular SMC in an additive manner
Protein kinase CK2 (formally casein kinase II) is an abundant
serine/threonine kinase located in different cellular compartments including
the plasma membrane (Faust and Montenarh,
2000). CK2 is overexpressed in proliferating tissues (for a
review, see Issinger, 1993
)
and CK2 activity has been correlated with cell proliferation
(Lebrin et al., 2001
).
Furthermore, a recent report has implicated CK2 in the mitogenic response of
human vascular SMC to recombinant UPA
(Dumler et al., 1999
). Based
on this evidence, we next investigated whether CK2 also plays a role in
mediating UPA-dependent cell proliferation following growth factor
stimulation. To this end, DNA synthesis assays in vascular SMC were performed
as described above using PDGF or bFGF stimulation in the absence or continuous
presence of the selective CK2 inhibitors, TBB or DRB (Shugar et al., 1994;
Sarno et al., 2001
). The data
obtained from these experiments demonstrated that blocking CK2 led to
inhibition of the PDGF- and bFGF-induced mitogenic response in vascular SMC.
TBB (4-10 µM) reduced BrdU incorporation in a dose-dependent manner up to
inhibition levels higher than 99% at a concentration of 10 µM, both in
PDGF- and bFGF-stimulated cells (Fig.
8). Significant, albeit much less pronounced inhibition was also
observed by the presence of DRB (40 µM DRB, PDGF: 64% reduction BrdU-LI;
bFGF: 63% reduction BrdU-LI, data not shown). Therefore, further experiments
were performed with TBB, which showed a reduction in DNA synthesis in vascular
SMC at concentrations reported as highly selective for inhibition of CK2
activity by others (Sarno et al.,
2001
). Concomitant addition of the UPA inhibitor
p-aminobenzamidine (100 and 200 µM) markedly enhanced the effect of TBB in
preventing the mitogenic activity of PDGF (12%, 45% and 57% inhibition at 4
µM TBB in the presence of 0, 100 and 200 µM p-aminobenzamidine,
respectively; P<0.005 for overall group differences;
Fig. 8A) and bFGF (37%, 89% and
84% inhibition at 4 µM TBB in the presence of 0, 100 and 200 µM
p-aminobenzamidine, respectively; P<0.005 for overall group
differences; Fig. 8B) in human
vascular SMC. In contrast, when HMW-UPA was added to the culture medium of the
TBB-treated cells, median levels of BrdU-uptake tended to recover to
approximately 90% of the median levels obtained in cells treated with PDGF or
bFGF alone (Table 4).
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Discussion |
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Upregulation of UPA expression by a diverse set of growth factors,
including bFGF and PDGF, has been described for a wide variety of carcinoma
cells (reviewed by Aguirre Ghiso et al.,
1999) as well as nontumor cells
(Fibbi et al., 1999
;
Miralles et al., 1998
;
Rusnati et al., 1997
). As yet,
however, only limited information is available on the effects of PDGF and bFGF
on UPA expression and secretion by vascular SMC. Using SMC of human umbilical
vein origin, we found that quiescent SMC constitutively expressed UPA.
However, mRNA and protein expression of UPA was several times increased in
proliferating cells upon stimulation with either PDGF or bFGF. Furthermore,
SMC stimulated with PDGF or bFGF secreted 5- to 11-times more UPA than control
cells. These observations are in line with the report by Clowes et al. showing
UPA overexpression in the neointima formed after vascular injury
(Clowes et al., 1990
). A
stimulatory effect of PDGF and bFGF on UPA antigen levels also has been
described by Wang and co-workers in SMC of human aortic origin
(Wang et al., 1997
). In
contrast, PDGF did not have an effect on UPA levels in murine vascular SMC
(Herbert et al., 1997
) and
neither PDGF nor bFGF affected UPA levels in cultured arterial SMC from
baboons (Kenagy and Clowes,
1995
). These divergent results are likely to reflect important
differences between species in the regulation of UPA expression in vascular
SMC.
In several types of tumor cells, the expression of UPA is associated with a
proliferative state (Aguirre Ghiso et al.,
1999). Synthesis of UPA has also been found to be highly regulated
during transition from the quiescent to the proliferative state in nontumor
murine (Grimaldi et al., 1986
)
and bovine (Zavizion et al.,
1998
) cells. Furthermore, UPA activity and DNA synthesis increase
in parallel in venular endothelial cells upon stimulation with bFGF
(Ziche et al., 1997
). Presta
et al. suggested that two independent pathways are involved in the mitogenic
and the UPA-inducing activity of bFGF
(Presta et al., 1992
). From
our studies we cannot exclude this possibility. However, our data suggest a
causal relationship between the expression of UPA in human vascular SMC
stimulated with bFGF and the ability of these cells to proliferate. Indeed, we
demonstrated that blocking endogenous UPA with a polyclonal antibody
significantly reduced DNA synthesis and cell growth in PDGF- and
bFGF-stimulated SMC. This decrease in DNA synthesis was already observed at
the time when quiescent SMC started to proliferate upon stimulation with the
growth factors. That UPA may act as a mediator of PDGF- and bFGF-induced
proliferation has already been suggested by observations in human hepatic
cells (Fibbi et al., 1999
).
Using SMC isolated from transgenic mice deficient in UPA, Herbert et al. also
provided evidence of a role for UPA in bFGF-induced cell growth
(Herbert et al., 1997
).
However, in contrast to our results, UPA did not appear to be involved in
PDGF-induced proliferation of the murine SMC.
An important finding in our study is that DNA synthesis and cell growth in
PDGF-and bFGF-stimulated vascular SMC were significantly abrogated in the
presence of a monoclonal antibody against the catalytic domain of UPA as well
as in the presence of the low-molecular-weight UPA inhibitor
p-aminobenzamidine. In contrast, lysine analogues that interfere with
plasminogen activation and the plasmin inhibitor aprotinin did not affect
mitogen-induced DNA synthesis. In line with these observations, only
functional UPA was found to act as a mitogen on human fibroblasts
(De Petro et al., 1994) and
melanoma cells (Kirchheimer et al.,
1989
). Despite the importance of a functional catalytic domain of
UPA, plasmin generation was shown not to be required for the mitogenic effects
of exogenous UPA (De Petro et al.,
1994
; Koopman et al.,
1998
; Herbert et al.,
1997
). The present study demonstrates a direct association between
the presence of functionally active UPA and the mitogenic effects of PDGF and
bFGF on human vascular SMC, which is independent of plasmin formation.
PDGF and bFGF, at concentrations that stimulate UPA expression, also
potentiate the expression and occupancy of its high affinity binding receptor
UPAR (Fibbi et al., 1999;
Reuning et al., 1994
). In
addition, the low molecular weight forms of UPA, containing the catalytic
domain but being devoid of the receptor binding region, do not exhibit
mitogenic activity (Koopman et al.,
1998
; Fischer et al.,
1998
; Kanse et al.,
1997
). These findings would support the view that the binding of
UPA to UPAR is essential for the induction of mitogenesis. However, currently
available information suggests that the importance of the UPA-UPAR interaction
is highly cell-type-specific with respect to cell proliferation. In our study,
blocking of either the UPA high affinity binding site on UPAR or the
N-terminal region of UPA with specific monoclonal antibodies did not alter the
mitogenic effect of PDGF and bFGF on vascular SMC. These findings suggest a
role for UPA in the proliferative response of human vascular SMC to these
growth factors that is independent of its high affinity binding to UPAR. In
agreement with our results, Carmeliet et al., using a model with targeted
disruption of the UPAR gene, demonstrated that binding of UPA to UPAR is not
required for medial and neointimal SMC proliferation in the injured vessel
wall (Carmeliet et al.,
1998
).
Our results do not exclude that binding of UPA to cell surface sites other
than UPAR is required for its role in the proliferative response of SMC to
PDGF and bFGF. For instance, melanoma cells have been found to show a
mitogenic response to exogenous UPA through a mechanism mediated by low
affinity binding of UPA to the cell surface rather than binding to UPAR
(Koopman et al., 1998).
Evidence for novel cell-surface UPA binding proteins has also recently been
reported for platelets (Jiang et al.,
1996
) and leukemic cell lines
(Longstaff et al., 1999
).
The precise molecular mechanisms involved in the UPA-induced mitogenesis
are currently unclear. UPA has been demonstrated to mediate cleavage and
activation of high molecular weight isoforms of vascular endothelial growth
factor (VEGF), which otherwise remain devoid of mitogenic activity
(Plouet et al., 1997). In a
series of experiments in which we pretreated PDGF and bFGF with human HMW-UPA,
the results did not provide any evidence that a similar proteolytic activation
was required for the induction of SMC proliferation by the recombinant forms
of PDGF and bFGF (data not shown).
Results by Dumler et al. endorse the notion that in human vascular cells
the mitogenic response to exogenous UPA occurs via activation of the protein
kinase CK2 (Dumler et al.,
1999). In the present study, the UPA-inhibitor p-aminobenzamidine
and 4,5,6,7-tetrabromobenzotriazole (TBB), a compound known as a selective
inhibitor of CK2 (Sarno et al.,
2001
), showed an additive effect in reducing DNA synthesis induced
by PDGF or bFGF. In turn, the addition of active human HMW-UPA tended to
counteract the decrease in DNA synthesis induced by the CK2 inhibitor.
Although preliminary, these findings raise the possibility that CK2 might also
play a role mediating UPA-dependent cell proliferation following growth factor
stimulation. Dumler et al. demonstrated that UPAR, CK2 and nucleolin are
colocalized on the cell surface of different cell types including vascular
SMC. The authors further suggested a role for this complex in the
proliferative response of vascular SMC to exogenous UPA
(Dumler et al., 1999
). Based
on our results, we can hypothesize that UPA-mediated cell proliferation
following PDGF and bFGF stimulation is likely to act through a
UPAR-independent CK2-activation mechanism, which still needs to be precisely
characterized. Interestingly, Bath et al. have provided evidence that UPA
induces tyrosine phosphorylation of a 78 kDa protein via a
proteolysis-dependent, but UPAR-independent mechanism. The authors have
suggested a link between activation of the 78 kDa protein and cell
proliferation in UPA-treated cells (Bhat et
al., 1999
).
In summary, we have demonstrated that UPA is an important mediator of the mitogenic effects of PDGF and bFGF on human vascular SMC. This proliferative activity of UPA in vascular SMC does not involve UPAR binding or plasmin generation, but does require a functional catalytic domain. According to these findings, the UPA catalytic domain may represent an attractive target for pharmacological interventions in atherogenesis or restenosis following angioplasty. Specific inhibition of the UPA catalytic domain may be beneficial not only in preventing plaque destabilization and rupture, but also in preventing SMC proliferation during neointima formation. Furthermore, inhibition of the catalytic site of UPA might by a way of antagonizing mitogenic effects of angiogenic cytokines such as bFGF (e.g. during tumor neovascularization).
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